Anti-angiogenic compositions and methods of use

ABSTRACT

The present invention provides compositions comprising an anti-angiogenic factor, and a polymeric carrier. Representative examples of anti-angiogenic factors include Anti-Invasive Factor, Retinoic acids and derivatives thereof, and taxol. Also provided are methods for embolizing blood vessels, and eliminating biliary, urethral, esophageal, and tracheal/bronchial obstructions.

CROSS-REFERENCE APPLICATIONS

This application claims the benefit of co-pending PCT applicationCA94/00373, filed Jul. 19, 1994. In addition, this application is acontinuation-in-part of pending U.S. patent application Ser. No.08/094,536, filed Jul. 19, 1993.

TECHNICAL FIELD

The present invention relates generally to compositions and methods fortreating cancer and other angiogenic-dependent diseases, and morespecifically, to compositions comprising anti-angiogenic factors andpolymeric carriers, stents which have been coated with suchcompositions, as well as method for utilizing these stents andcompositions.

BACKGROUND OF THE INVENTION

Angiogenesis-dependent diseases (i.e., those diseases which require orinduce vascular growth) represent a significant portion of all diseasesfor which medical treatment is sought. For example, cancer is the secondleading cause of death in the United States, and accounts for overone-fifth of the total mortality. Briefly, cancer is characterized bythe uncontrolled division of a population of cells which, mosttypically, leads to the formation of one or more tumors. Such tumors arealso characterized by the ingrowth of vasculature which provide variousfactors that permit continued tumor growth. Although cancer is generallymore readily diagnosed than in the past, many forms, even if detectedearly, are still incurable.

A variety of methods are presently utilized to treat cancer, includingfor example, various surgical procedures. If treated with surgery alonehowever, many patients (particularly those with certain types of cancer,such as breast, brain, colon and hepatic cancer) will experiencerecurrence of the cancer. Therefore, in addition to surgery, manycancers are also treated with a combination of therapies involvingcytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine,cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy. Onedifficulty with this approach, however, is that radiotherapeutic andchemotherapeutic agents are toxic to normal tissues, and often createlife-threatening side effects. In addition, these approaches often haveextremely high failure/remission rates.

In addition to surgical, chemo- and radiation therapies, others haveattempted to utilize an individual's own immune system in order toeliminate cancerous cells. For example, some have suggested the use ofbacterial or viral components as adjuvants in order to stimulate theimmune system to destroy tumor cells. (See generally “Principles ofCancer Biotherapy,” Oldham (ed.), Raven Press, New York, 1987.) Suchagents have generally been useful as adjuvants and as nonspecificstimulants in animal tumor models, but have not as of yet proved to begenerally effective in humans.

Lymphokines have also been utilized in the treatment of cancer. Briefly,lymphokines are secreted by a variety of cells, and generally have aneffect on specific cells in the generation of an immune response.Examples of lymphokines include Interleukins (IL)-1, -2, -3, and -4, aswell as colony stimulating factors such as G-CSF, GM-CSF, and M-CSF.Recently, one group has utilized IL-2 to stimulate peripheral bloodcells in order to expand and produce large quantities of cells which arecytotoxic to tumor cells (Rosenberg et al., N. Engl. J. Med.313:1485-1492, 1985).

Others have suggested the use of antibodies in the treatment of cancer.Briefly, antibodies may be developed which recognize certain cellsurface antigens that are either unique, or more prevalent on cancercells compared to normal cells. These antibodies, or “magic bullets,”may be utilized either alone or conjugated with a toxin in order tospecifically target and kill tumor cells (Dillman, “Antibody Therapy,”Principles of Cancer Biotherapy, Oldham (ed.), Raven Press, Ltd., NewYork, 1987). However, one difficulty is that most monoclonal antibodiesare of murine origin, and thus hypersensitivity against the murineantibody may limit its efficacy, particularly after repeated therapies.Common side effects include fever, sweats and chills, skin rashes,arthritis, and nerve palsies.

One additional difficulty of present methods is that local recurrenceand local disease control remains a major challenge in the treatment ofmalignancy. In particular, a total of 630,000 patients annually (in theU.S.) have localized disease (no evidence of distant metastatic spread)at the time of presentation; this represents 64% of al those patientsdiagnosed with malignancy (this does not include nonmelanoma skin canceror carcinoma in situ). For the vast majority of these patients, surgicalresection of the disease represents the greatest chance for a cure andindeed 428,000 will be cured after the initial treatment—428,000.Unfortunately, 202,000 (or 32% of all patients with localized disease)will relapse after the initial treatment. Of those who relapse, thenumber who will relapse due to local recurrence of the disease amountsto 133,000 patients annually (or 21% of all those with localizeddisease). The number who will relapse due to distant metastases of thedisease is 68,000 patients annually (11% of all those with localizeddisease). Another 102,139 patients annually will die as a direct resultof an inability to control the local growth of the disease.

Nowhere is this problem more evident than in breast cancer, whichaffects 186,000 women annually in the U.S. and whose mortality rate hasremained unchanged for 50 years. Surgical resection of the diseasethrough radical mastectomy, modified radical mastectomy, or lumpectomyremains the mainstay of treatment for this condition. Unfortunately, 39%of those treated with lumpectomy alone will develop a recurrence of thedisease, and surprisingly, so will 25% of those in which the resectionmargin is found to be clear of tumor histologically. As many as 90% ofthese local recurrences will occur within 2 cm of the previous excisionsite.

Similarly, in 1991, over 113,000 deaths and 238,600 new cases of livermetastasis were reported in North America alone. The mean survival timefor patients with liver metastases is only 6.6 months once liver lesionshave developed. Non-surgical treatment for hepatic metastases includesystemic chemotherapy, radiation, chemoembolization, hepatic arterialchemotherapy, and intraarterial radiation. However, despite evidencethat such treatments can transiently decrease the size of the hepaticlesions (e.g., systemic chemotherapy and hepatic arterial chemotherapyinitially reduces lesions in 15-20%, and 80% of patients, respectively),the lesions invariably reoccur. Surgical resection of liver metastasesrepresents the only possibility for a cure, but such a procedure ispossible in only 5% of patients with metastases, and in only 15-20% ofpatients with primary hepatic cancer.

One method that has been attempted for the treatment of tumors withlimited success is therapeutic embolization. Briefly, blood vesselswhich nourish a tumor are deliberately blocked by injection of anembolic material into the vessels. A variety of materials have beenattempted in this regard, including autologous substances such as fat,blood clot, and chopped muscle fragments, as well as artificialmaterials such as wool, cotton, steel balls, plastic or glass beads,tantalum powder, silicone compounds, radioactive particles, sterileabsorbable gelatin sponge (Sterispon, Gelfoam), oxidized cellulose(Oxycel), steel coils, alcohol, lyophilized human dura mater (Lyodura),microfibrillar collagen (Avitene), collagen fibrils (Tachotop),polyvinyl alcohol sponge (PVA; Ivalon), Barium-impregnated siliconspheres (Biss) and detachable balloons. The size of liver metastases maybe temporarily decreased utilizing such methods, but tumors typicallyrespond by causing the growth of new blood vessels into the tumor.

A related problem to tumor formation is the development of cancerousblockages which inhibit the flow of material through body passageways,such as the bile ducts, trachea, esophagus, vasculature and urethra. Onedevice, the stent, has been developed in order to hold open passagewayswhich have been blocked by tumors or other substances. Representativeexamples of common stents include the Wallstent, Strecker stent,Gianturco stent, and the Palmaz stent. The major problem with stents,however, is that they do not prevent the ingrowth of tumor orinflammatory material through the interstices of the stent. If thismaterial reaches the inside of a stent and compromises the stent lumen,it may result in blockage of the body passageway into which it has beeninserted. In addition, presence of a stent in the body may inducereactive or inflammatory tissue (e.g., blood vessels, fibroblasts, whiteblood cells) to enter the stent lumen, resulting in partial or completeclosure of the stent.

The present invention provides compositions and methods suitable fortreating cancers, as well as other non-tumorigenicangiogenesis-dependent diseases, and further provides other relatedadvantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides anti-angiogeniccompositions, as well as methods and devices which utilize suchcompositions for the treatment of cancer and otherangiogenesis-dependent diseases. Within one aspect of the presentinvention, compositions are provided (anti-angiogenic compositions)comprising (a) an anti-angiogenic factor and (b) a polymeric carrier. Awide variety of molecules may be utilized within the scope of thepresent invention as anti-angiogenic factors, including for exampleAnti-Invasive Factor, retinoic acids and their derivatives, paclitaxelincluding analogues and derivatives thereof, Suramin, Tissue Inhibitorof Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1 and Plasminogen Activator Inhibitor-2,and lighter “d group” transition metals. Similarly, a wide variety ofpolymeric carriers may be utilized, representative examples of whichinclude poly (ethylene-vinyl acetate) (40% cross-linked), poly(D,L-lactic acid) oligomers and polymers, poly (L-lactic acid) oligomersand polymers, poly (glycolic acid), copolymers of lactic acid andglycolic acid, poly (caprolactone), poly (valerolactone), poly(anhydrides), copolymers of poly (caprolactone) or poly (lactic acid)with polyethylene glycol, and blends thereof.

Within certain preferred embodiments, the compositions comprise acompound which disrupts microtubule function, such as, for example,paclitaxel, estramustine, colchicine, methotrexate, curacin-A,epothilone, vinblastine or tBCEV. Within other preferred embodiments,the compositions comprise a polymeric carrier and a lighter d groputransition metal (e.g., a vanadium species, molybdenum species, tungstenspecies, titanium species, niobium species or tantalum species) whichinhibits the formation of new blood vessels.

Within one embodiment of the invention, the composition has an averagesize of 15 to 200 μm, within other embodiments, the polymeric carrier ofthe composition has a molecular weight ranging from less than 1,000daltons to greater than 200,000 to 300,000 daltons. Within yet otherembodiments, the compositions provided herein may be formed into filmswith a thickness of betweem 100 μm and 2 mm, or thermologically activecompositions which are liquid at one temperature (e.g., above 45° C.)and solid or semi-solid at another (e.g., 37° C.).

Within another aspect of the present invention methods for embolizing ablood vessel are provided, comprising the step of delivering into thevessel a therapeutically effective amount of an anti-angiogeniccomposition (as described above), such that the blood vessel iseffectively occluded. Within one embodiment, the anti-angiogeniccomposition is delivered to a blood vessel which nourishes a tumor.

Within yet another aspect of the present invention, stents are providedcomprising a generally tubular structure, the surface being coated withone or more anti-angiogenic compositions. Within other aspects of thepresent invention, methods are provided for expanding the lumen of abody passageway, comprising inserting a stent into the passageway, thestent having a generally tubular structure, the surface of the structurebeing coated with an anti-angiogenic composition as described above,such that the passageway is expanded. Within various embodiments of theinvention, methods are provided for eliminating biliary obstructions,comprising inserting a biliary stent into a biliary passageway; foreliminating urethral obstructions, comprising inserting a urethral stentinto a urethra; for eliminating esophageal obstructions, comprisinginserting an esophageal stent into an esophagus; and for eliminatingtracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi. In each of theseembodiments, the stent has a generally tubular structure, the surface ofwhich is coated with an anti-angiogenic composition as described above.

Within another aspect of the present invention, methods are provided fortreating tumor excision sites, comprising administering ananti-angiogenic composition as described above to the resection marginsof a tumor subsequent to excision, such that the local recurrence ofcancer and the formation of new blood vessels at the site is inhibited.Within yet another aspect of the invention, methods for treating cornealneovascularization are provided, comprising the step of administering toa patient a therapeutically effective amount of an anti-angiogeniccomposition as described above to the cornea, such that the formation ofblood vessels is inhibited. Within one embodiment, the anti-angiogeniccomposition further comprises a topical corticosteroid.

Within another aspect of the present invention, methods are provided forinhibiting angiogenesis in patients with non-tumorigenic,angiogenesis-dependent diseases, comprising administering to a patient atherapeutically effective amount of paclitaxel to a patient with anon-tumorigenic angiogenesis-dependent disease, such that the formationof new blood vessels is inhibited. Within other aspects, methods areprovided for embolizing blood vessels in non-tumorigenic,angiogenesis-dependent diseases, comprising delivering to the vessel atherapeutically effective amount of a composition comprising paclitaxel,such that the blood vessel is effectively occluded.

Within yet other aspects of the present invention, methods are providedfor expanding the lumen of a body passageway, comprising inserting astent into the passageway, the stent having a generally tubularstructure, the surface of the structure being coated with a compositioncomprising paclitaxel, such that the passageway is expanded. Withinvarious embodiments of the invention, methods are provided foreliminating biliary obstructions, comprising inserting a biliary stentinto a biliary passageway; for eliminating urethral obstructions,comprising inserting a urethral stent into a urethra; for eliminatingesophageal obstructions, comprising inserting an esophageal stent intoan esophagus; and for eliminating tracheal/bronchial obstructions,comprising inserting a tracheal/bronchial stent into the trachea orbronchi. Within each of these embodiments the stent has a generallytubular structure, the surface of the structure being coated with acomposition comprising paclitaxel.

Within another aspect of the present invention, methods are provided fortreating a tumor excision site, comprising administering a compositioncomprising paclitaxel to the resection margin of a tumor subsequent toexcision, such that the local recurrence of cancer and the formation ofnew blood vessels at the site is inhibited. Within other aspects,methods are provided for treating neovascular diseases of the eye,comprising administering to a patient a therapeutically effective amountof an anti-angiogenic factor (such as a compound which disruptsmicrotubule function) to the eye, such that the formation of new vesselsis inhibited.

Within other aspects of the present invention, methods are provided fortreating inflammatory arthritis, comprising administering to a patient atherapeutically effective amount of an anti-angiogenic factor (such as acompound which disrupts microtubule function), or a compositioncomprising an anti-angiogenic factor and a polymeric carrier to a joint.Within preferred embodiments, the anti-angiogenic factor may be acompound which disrupts microtubule function such as paclitaxel, or anelement from the lighter ‘d group’ transition metals, such as a vanadiumspecies.

Within yet another aspect of the invention, pharmaceutical products areprovided, comprising (a) a compound which disrupts microtubule function,in a container, and (b) a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of a compound which disrupts microtubule function, for human orveterinary administration to treat non-tumorigenicangiogenesis-dependent diseases such as, for example, inflammatoryarthritis or neovascular diseases of the eye. Briefly, Federal Lawrequires that the use of a pharmaceutical agent in the therapy of humansbe approved by an agency of the Federal government. Responsibility forenforcement (in the United States) is with the Food and DrugAdministration, which issues appropriate regulations for securing suchapproval, detailed in 21 U.S.C. §§ 301-392. Regulation for biologicalmaterials comprising products made from the tissues of animals, is alsoprovided under 42 U.S.C. § 262. Similar approval is required by mostcountries, although, regulations may vary from country to country.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures, devices or compositions, andare therefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph which shows a shell-less egg culture on day 6.FIG. 1B is a digitized computer-displayed image taken with astereomicroscope of living, unstained capillaries (1040×). FIG. 1C is aphotograph of a corrosion casting which shows CAM microvasculature thatare fed by larger, underlying vessels (arrows; 1300×). FIG. 1D is aphotograph which depicts a 0.5 mm thick plastic section cut transverselythrough the CAM, and recorded at the light microscope level. Thisphotograph shows the composition of the CAM, including an outerdouble-layered ectoderm (Ec), a mesoderm (M) containing capillaries(arrows) and scattered adventitila cells, and a single layered endoderm(En) (400×). FIG. 1E is a photograph at the electron microscope level(3500×) wherein typical capillary structure is presented showingthin-walled endothelial cells (arrowheads) and an associated pericyte.

FIGS. 2A, 2B, 2C and 2D are a series of digitized images of fourdifferent, unstained CAMs taken after a 48 hour exposure to digitizedimages of four different living, unstained CAMs were taken after a 48 hexposure to 10μpaclitaxel per 10 ml of methylcellulose. The transparentmethylcellulose disk (*) containing paclitaxel is present on each CAMand is positioned over a singular avascular zone (A) with surroundingeblood islands (Is). These avascular areas extend beyond the disk andtypically have a diameter of 6 mm. FIG. 2D illustrates the typical“elbowing” effect (arrowheads) of both small and large vessels beingredirected away from the periphery of the avascular zone.

FIG. 3A is a photograph (Mag=400×) which shows just peripheral to theavascular zone, that capillaries (arrowheads) exhibit numerousendothelial cells arrested in mitosis. Ectoderm (Ec); Mesoderm (M);Endoderm (En). FIG. 3B (Mag=400×) shows that within the avascular zoneproper the typical capillary structure has been eliminated and there arenumerous extravasated blood cells (arrowheads). FIG. 3C (Mag=400×) showsthat in the central area of the avascular zone, red blood cells aredispersed throughout the mesoderm.

FIGS. 3A, 3B and 3C are a series of photographs of 0.5 mm thick plasticsections transversely cut through a paclitaxel-treated CAM at threedifferent locations within the avascular zone.

FIGS. 4A, 4B and 4C are series of electron micrographs which were takenfrom locations similar to that of FIGS. 3A, 3B and 3C (respectively)above.

FIG. 4A (Mag=2,200×) shows a small capillary lying subjacent to theectodermal layer (Ec) possessing three endothelial cells arrested inmitosis (*). Several other cell types in both the ectodern and mesodermare also arrested in mitosis. FIG. 4B (Mag=2,800×) shows the earlyavascular phase contains extravasated blood cells subjacent to theectoderm; these blood cells are intermixed with presumptive endothelialcells (*) and their processes. Degrative cellular vacuoles (arrowhead).FIG. 4C (Mag=2,800×) shows that in response to paclitaxel, theecto-mesodermal interface has become populated with cells in variousstages of degradation containing dense vacuoles and granules(arrowheads).

FIG. 5 is a bar graph which depicts the size distribution ofmicrospheres by number (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

FIG. 6 is a bar graph which depicts the size distribution ofmicrospheres by weight (5% poly (ethylene-vinyl acetate) with 10 mgsodium suramin into 5% PVA).

FIG. 7 is a graph which depicts the weight of encapsulation of SodiumSuramin in 50 mg poly (ethylene-vinyl acetate).

FIG. 8 is a graph which depicts the percent of encapsulation of SodiumSuramin in 50 mg poly (ethylene-vinyl acetate).

FIG. 9 is a bar graph which depicts the size distribution by weight of5% ELVAX microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 10 is a bar graph which depicts the size distribution by weight of5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 11 is a bar graph which depicts the size distribution by number of5% microspheres containing 10 mg sodium suramin made in 5% PVAcontaining 10% NaCl.

FIG. 12 is a line graph which depicts the time course of sodium suraminrelease.

FIG. 13 is an illustration of a representative embodiment of hepatictumor embolization.

FIG. 14 is an illustration of the insertion of a representative stentcoated with an anti-angiogenic composition.

FIG. 15 A is a graph which shows the effect of the EVA:PLA polymer blendratio upon aggregation of microspheres. FIG. 15B is a scanning electronmicrograph which shows the size of “small” microspheres. FIG. 15C (whichincludes a magnified inset—labelled “15C-inset”) is a scanning electronmicrograph which shows the size of “large” microspheres. FIG. 15D is agraph which depicts the time course of in vitro paclitaxel release from0.6% w/v paclitaxel-loaded 50:50 EVA:PLA polymer blend microspheres intophosphate buffered saline (pH 7.4) at 37° C. Open circles are “small”sized microspheres, and closed circles are “large” sized microspheres.FIG. 15F is a photograph of a CAM which shows the results of paclitaxelrelease by microspheres (“MS”). FIG. 15F is a photograph similar to thatof 15E at increased magnification.

FIG. 16 is a graph which shows release rate profiles frompolycaprolactone microspheres containing 1%, 2%, 5% or 10% paclitaxelinto phosphate buffered saline at 37° C. FIG. 16B is a photograph whichshows a CAM treated with control microspheres. FIG. 16C is a photographwhich shows a CAM treated with 5% paclitaxel loaded microspheres.

FIGS. 17A and 17B, respectively, are two graphs which show the releaseof paclitaxel from EVA films, and the percent paclitaxel remaining inthose same films over time. FIG. 17C is a graph which shows the swellingof EVA/F127 films with no paclitaxel over time. FIG. 17D is a graphwhich shows the swelling of EVA/Span 80 films with no paclitaxel overtime. FIG. 17E is a graph which depicts a stress vs. strain curve forvarious EVA/F127 blends.

FIGS. 18A and 18B are two graphs which show the melting point ofPCL/MePEG polymer blends as a function of % MePEG in the formulation(18A), and the percent increase in time needed for PCL paste at 60° C.to being to solidify as a function of the amount of MePEG in theformulation (18B). FIG. 18C is a graph which depicts the softness ofvarying PCL/MePEG polymer blends. FIG. 18D is a graph which shows thepercent weight change over time for polymer blends of various MePEGconcentrations. FIG. 18E is a graph which depicts the rate of paclitaxelrelease over time from various polymer blends loaded with 1% paclitaxel.FIGS. 18F and 18G are graphs which depict the effect of varyingquantities of paclitaxel on the total amount of paclitaxel released froma 20% MePEG/PCL blend. FIG. 18H is a graph which depicts the effect ofMePEG on the tensile strength of a MePEG/PCL polymer.

FIG. 19A is a photograph which shows control (unloaded) thermopaste on aCAM. Note that both large vessels and small vessels (capillaries) arefound immediately adjacent to the paste. Blood flow in the area aroundand under the paste is unaffected. FIG. 19B is a photograph of 20%paclitaxel-loaded thermopaste on a CAM. Note the disruption of thevasculature when compared to the surrounding tissues. The drug loadedpaste has blocked the growth of the capillaries, caused regression ofthe larger vessels, and created a region of avascularity on the CAMassay. FIG. 19C is a photograph of 0.5% paclitaxel-loaded thermopaste ona CAM (Mag.—40×). Briefly, the paclitaxel-loaded thermopaste diskinduced an avascular zone measuring 6 mm in diameter on the CAM. Thisavascular region was induced by blocking new capillary growth andoccluding, disrupting, and regressing the existing blood vessels foundwithin the treated region. FIG. 19D is a photograph of control(unloaded) Thermopaste on a CAM. Briefly, after a 2 day exposure, theblood vessel organization of the CAM (Mag=50×) treated with the controlpaste shows normal blood vessel organization. Functional vessels arelocated immediately adjacent to the unloaded paste.

FIGS. 20A and 20B are two photographs of a CAM having a tumor treatedwith control (unloaded) thermopaste. Briefly, in FIG. 20 A the centralwhite mass is the tumor tissue. Note the abundance of blood vesselsentering the tumor from the CAM in all directions. The tumor induces theingrowth of the host vasculature through the production of “angiogenicfactors.” The tumor tissue expands distally along the blood vesselswhich supply it. FIG. 20B is an underside view of the CAM shown in 20A.Briefly, this view demonstrates the radial appearance of the bloodvessels which enter the tumor like the spokes of a wheel. Note that theblood vessel density is greater in the vicinity of the tumor than it isin the surrounding normal CAM tissue. FIGS. 20C and 20D are twophotographs of a CAM having a tumor treated with 20% paclitaxel-loadedthermopaste. Briefly, in FIG. 20C the central white mass is the tumortissue. Note the paucity of blood vessels in the vicinity of the tumortissue. The sustained release of the angiogenesis inhibitor is capableof overcoming the angiogenic stimulus produced by the tumor. The tumoritself is poorly vascularized and is progressively decreasing in size.FIG. 20D is taken from the underside of the CAM shown in 20C, anddemonstrates the disruption of blood flow into the tumor when comparedto control tumor tissue. Note that the blood vessel density is reducedin the vicinity of the tumor and is sparser than that of the normalsurrounding CAM tissue.

FIG. 21A is a graph which shows the effect of paclitaxel/PCL on tumorgrowth. FIGS. 21B and 21C are two photographs which show the effect ofcontrol, 10%, and 20% paclitaxel-loaded thermopaste on tumor growth.

FIG. 22A is a photograph of synovium from a PBS injected joint. FIG. 22Bis a photograph of synovium from a microsphere injected joint. FIG. 22Cis a photograph of cartilage from joints injected with PBS, and FIG. 22Dis a photograph of cartilage from joints injected with microspheres.

FIG. 23A is a photograph of a 0.3% Paclitaxel Ophthalmic Drop Suspensionon a CAM (Mag.=32×). The plastic ring was used to localize the drugtreatment to the CAM. Note the lack of blood vessels located within andimmediately adjacent to the ring. The functional blood vessels borderingthe avascular zone are defined by their “elbowing” morphology away formthe drug source. FIG. 23B is a photograph of a control (unloaded)Ophthalmic Drop Suspension on a CAM (Mag=32×). Note the normalorganization of the CAM blood vessels and the abundance of functionalvessels located within the ring.

FIG. 24A is a photograph of a 2.5% Paclitaxel-Loaded Stent Coating(Mag=26×). Briefly, the blood vessels surrounding the avascular zone aremorphologically redirected away from the paclitaxel source; thisproduces an avascular zone which can measure up to 6 mm in diameter. Thedisrupted vascular remnants which represent vascular regression can beseen within the avascular zone. FIG. 24B is a control (unloaded) StentCoating (Mag=26×). Briefly, the blood vessels of the CAM are foundimmediately adjacent to the stent and do not illustrate anymorphological alterations.

FIG. 25 is a photograph of a control stent. Briefly, this image showsthe longitudinal orientation of a nylon stent incorporated withingliosarcoma tissue of the rat liver. Ingrowth within the nylon stent isevident.

FIG. 26 is a photograph of a control stent. Briefly, this image alsoillustrates tumor ingrowth within the lumen of the nylon stent.

FIG. 27 is a photograph of a lung. Briefly, in addition to large livertumors, metastasis to the lung is common. Such metastases are evident bythe presence of small white lobules seen throughout the lung.

FIG. 28A is a photograph of Suramin and Cortisone Acetate on a CAM(Mag=8×). Briefly, this image shows an avascular zone treated with 20 μgof suramin and 70 μg of cortisone acetate in 0.5% methylcellulose. Notethe blood vessels located at the periphery of the avascular zone whichare being redirected away from the drug source. FIG. 28B is a photographwhich shows the vascular detail of the effected region at a highermagnification (Mag=20×). Note the avascular regions and the typical“elbowing” effect of the blood vessels bordering the avascular zone.

FIG. 29A is a graph which shows the chemiluminescence response ofneutrophils (5×10⁶ cells/ml) to plasma opsonized CPPD crystals (50mg/ml). Effect of paclitaxel at (∘) no paclitaxel, (●) 4.5 μM, (Δ,) 14μM, (▴) 28 μM, (□) 46 μM; n=3. FIG. 29B is a graph which shows the timecourse concentration dependence of paclitaxel inhibition of plasmaopsonized CPPD crystal induced neutrophil chemiluminescence.

FIG. 30A is a graph which shows superoxide anion production byneutrophils (5×10⁶ cells/ml) in response to plasma opsonized CPPDcrystals (50 mg/ml). Effect of paclitaxel at (∘) no paclitaxel, (●) 28μM,. (Δ) Control (cells alone); n=3. FIG. 30B is a graphic which showsthe time course concentration dependence of paclitaxel inhibition ofplasma opsonized CPPD crystal induced neutrophil superoxide anionproduction; n 3.

FIG. 31A is a graph which shows the chemiluminescence response ofneutrophils (5×10⁶ cells/ml) in response to plasma opsonized zymozan (1mg/ml). Effect of paclitaxel at (∘) no drug, (●) 28 μM; n=3. FIG. 31B isa graph which shows plasma opsonized zymosan induced neutrophilsuperoxide anion production. Effect of paclitaxel at (∘) no paclitaxel,(●) 28 μM, (Δ) Control (cells alone); n 3.

FIG. 32A is a graph which shows myeloperoxidase release from neutrophils(5×10⁶ cells/ml) in response to plasma opsonized CPPD crystals (50mg/ml). Effect of paclitaxel at (∘) no paclitaxel, (●) 28 μM. (Δ)Control (cells alone), (▴) Control (cells with paclitaxel at 28 μM);n=3.

FIG. 32B is a graph which shows the concentration dependence ofpaclitaxel inhibition of myeloperoxidase release from neutrophils inresponse to plasma opsonized CPPD crystals; n 3.

FIG. 33 is a graph which shows lysozyme release from neutrophils(5×10⁶/ml) in response to plasma opsonized CPPD crystals (50 mg/ml).Effect of paclitaxel at (∘) no paclitaxel, (●) 28 μM, (Δ) Control (cellsalone), (▴) Control (cells and paclitaxel at 28 μM); n 3.

FIG. 34 is a graph which depicts proliferation of synoviocytes atvarious concentrations of paclitaxel.

FIG. 35 is a bar graph which depicts the cytotoxicity of paclitaxel atvarious concentrations to proliferating synoviocytes.

FIGS. 36A, 36B, and 36C are photographs of a series of gels which showthe effect of various concentrations of paclitaxel on c-FOS expression.

FIGS. 37A and 37B are photographs of a series of gels which show theeffect of various concentrations of paclitaxel on collagenaseexpression.

FIG. 38 is a bar graph which depicts the effects of paclitaxel onviability of normal chondrocytes in vitro.

FIG. 39 is a graph which shows the percentage of paclitaxel releasebased upon gelatinized-paclitaxel of either a large (7200 μm) or small(2100 μm) size.

FIG. 40 is a graph which shows the effect of gelatin and/or sodiumchloride on the release of paclitaxel from PCL.

FIG. 41 is a graph which shows the release of paclitaxel fromPDLLA-PEG-PDLLA cylinders containing 20% paclitaxel.

FIG. 42A is a graph which depicts the time course of paclitaxel releasefrom 2.5 mg pellets of PCL. FIG. 42B is a graph which shows the percentpaclitaxel remaining in the pellet, over time.

FIG. 43A is a graph which shows the effect of MePEG on paclitaxelrelease from PCL paste leaded with 20% paclitaxel. FIG. 43B is a graphwhich shows the percent paclitaxel remaining in the pellet, over time.

FIGS. 44A and 44B are graphs which show the effect of variousconcentrations of MePEG in PCL in terms of melting point (44A) and timeto solidify (44B).

FIG. 45 is a graph which shows the effect of MePEG incorporation intoPCL on the tensile strength and time to fail of the polymer.

FIG. 46 is a graph which shows the effect of irradiation on paclitaxelrelease.

FIGS. 47A, B, C, D and E show the effect of MTX release from PCL overtime.

FIG. 48 is a graph of particle diameter (μm) determined by a Coulter®LS130 Particle Size Analysis.

FIG. 49 is a graph of particle diameter (μm) determined by a Coulter®LS130 Particle Size Analysis.

FIG. 50 is a graph which shows paclitaxel release from various polymericformulations.

FIG. 51 is a graph which shows the effect of plasma opsonization ofpolymeric microspheres on the chemiluminescence response of neutrophils(20 mg/ml microspheres in 0.5 ml of cells (conc. 5×10⁶ cells/ml) to PCLmicrospheres.

FIG. 52 is a graph which shows the effect of precoating plasma ±2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PCL microspheres

FIG. 53 is a graph which shows the effect of precoating plasma ±2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PMMA microspheres

FIG. 54 is a graph which shows the effect of precoating plasma ±2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to PLA microspheres

FIG. 55 is a graph which shows the effect of precoating plasma ±2%pluronic F127 on the chemiluminescence response of neutrophils (5×10⁶cells/ml) to EVA:PLA microspheres

FIG. 56 is a graph which shows the effect of precoating IgG (2 mg/ml),or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence responseof neutrophils to PCL microspheres.

FIG. 57 is a graph which shows the effect of precoating IgG (2 mg/ml),or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence responseof neutrophils to PMMA microspheres.

FIG. 58 is a graph which shows the effect of precoating IgG (2 mg/ml),or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence responseof neutrophils to PVA microspheres.

FIG. 59 is a graph which shows the effect of precoating IgG (2 mg/ml),or 2% pluronic F127 then IgG (2 mg/ml) on the chemiluminescence responseof neutrophils to EVA:PLA microspheres.

FIG. 60 is a photograph of 10% methotrexate (“MTX”) loaded microspheresmade from a 50:50 ratio of PLA:GA (IV 0.78).

FIG. 61 is a graph which depicts the release of 10% loaded vanadylsulfate from PCL.

FIG. 62 is a photograph of hyaluronic acid microspheres containingvanadium sulfate.

FIG. 63A is a graph which depicts the release of organic vanadate fromPCL. FIG. 63B depicts the percentage of organic vanadate remaining overa time course.

FIG. 64 is a photograph showing poly D,L, lactic acid microspherescontaining organic vanadate.

FIGS. 65A and 65B are photographs of control (uncoated) stents whichshow typical epithelial ingrowth seen at both 8 weeks (A) and at 16weeks (B). Indentations of the stent tines (t) and narrowing of thelumen (lu) are shown. There is progressive epithelial overgrowth of thestent surface over this time by fibrous and inflammatory tissue.

FIGS. 66A, 66B, 66C, and 66D are a series of photographs which showcontrol and paclitaxel-coated biliary stents. FIG. 66A illustrates theobliteration of the stent lumen by the process of benign epithelialovergrowth. At higher magnification (66B), the fibrous and inflammatorytissue is evident with little luminal space remaining. Thepaclitaxel-treated biliary duct remains patent (66C). At highermagnification, normal biliary tract epithelium is present with onlyminimal alteration of the mucosal lining by the coated stent tines (t).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods and compositionswhich utilize anti-angiogenic factors. Briefly, within the context ofthe present invention, anti-angiogenic factors should be understood toinclude any protein, peptide, chemical, or other molecule, which acts toinhibit vascular growth. A variety of methods may be readily utilized todetermine the anti-angiogenic activity of a given factor, including forexample, chick chorioallantoic membrane (“CAM”) assays. Briefly, asdescribed in more detail below in Examples 2A and 2C, a portion of theshell from a freshly fertilized chicken egg is removed, and a methylcellulose disk containing a sample of the anti-angiogenic factor to betested is placed on the membrane. After several days (e.g., 48 hours),inhibition of vascular growth by the sample to be tested may be readilydetermined by visualization of the chick chorioallantoic membrane in theregion surrounding the methyl cellulose disk. Inhibition of vasculargrowth may also be determined quantitatively, for example, bydetermining the number and size of blood vessels surrounding the methylcellulose disk, as compared to a control methyl cellulose disk. Althoughanti-angiogenic factors as described herein are considered to inhibitthe formation of new blood vessels if they do so in merely astatistically significant manner, as compared to a control, withinpreferred aspects such anti-angiogenic factors will completely inhibitthe formation of new blood vessels, as well as reduce the size andnumber of previously existing vessels.

In addition to the CAM assay described above, a variety of other assaysmay also be utilized to determine the efficacy of anti-angiogenicfactors in vivo, including for example, mouse models which have beendeveloped for this purpose (see Roberston et al., Cancer. Res.51:1339-1344, 1991). In addition, a variety of representative in vivoassays relating to various aspects of the inventions described hereinhave also been described in more detail below in Examples 5 to 7, and 17to 19.

As noted above, the present invention provides compositions comprisingan anti-angiogenic factor, and a polymeric carrier. Briefly, a widevariety of anti-angiogenic factors may be readily utilized within thecontext of the present invention. Representative examples includeAnti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel,Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1, PlasminogenActivator Inhibitor-2, and various forms of the lighter “d group”transition metals. These and other anti-angiogenic factors will bediscussed in more detail below.

Briefly, Anti-Invasive Factor, or “AIF” which is prepared from extractsof cartilage, contains constituents which are responsible for inhibitingthe growth of new blood vessels. These constituents comprise a family of7 low molecular weight proteins (<50,000 daltons) (Kuettner and Pauli,“Inhibition of neovascularization by a cartilage factor” in Developmentof the Vascular System, Pitman Books (CIBA Foundation Symposium 100),pp. 163-173, 1983), including a variety of proteins which haveinhibitory effects against a variety of proteases (Eisentein et al, Am.J. Pathol. 81:337-346, 1975; Langer et al., Science 193:70-72, 1976; andHorton et al., Science 199:1342-1345, 1978). AIF suitable for use withinthe present invention may be readily prepared utilizing techniques knownin the art (e.g., Eisentein et al, supra; Kuettner and Pauli, supra; andLanger et al., supra). Purified constituents of AIF such asCartilage-Derived Inhibitor (“CDI”) (see Moses et al., Science248:1408-1410, 1990) may also be readily prepared and utilized withinthe context of the present invention.

Retinoic acids alter the metabolism of extracellular matrix components,resulting in the inhibition of angiogenesis. Addition of prolineanalogs, angiostatic steroids, or heparin may be utilized in order tosynergistically increase the anti-angiogenic effect of transretinoicacid. Retinoic acid, as well as derivatives thereof which may also beutilized in the context of the present invention, may be readilyobtained from commercial sources, including for example, Sigma ChemicalCo. (# R2625).

Paclitaxel is a highly derivatized diterpenoid (Wani et al., J. Am.Chem. Soc. 93:2325, 1971) which has been obtained from the harvested anddried bark of Taxus brevifolia (Pacific Yew.) and Taxomyces Andreanaeand Endophytic Fungus of the Pacific Yew (Stierle et al., Science60:214-216, 1993). Generally, paclitaxel acts to stabilize microtubularstructures by binding tubulin to form abnormal mitotic spindles.“Paclitaxel” (which should be understood herein to include analogues andderivatives such as, for example, TAXOL®, TAXOTERE®, 10-desacetylanalogues of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxy carbonylanalogues of paclitaxel) may be readily prepared utilizing techniquesknown to those skilled in the art (see also WO 94/07882, WO 94/07881, WO94/07880, WO 94/07876, WO 93/23555, WO 93/10076, U.S. Pat. Nos.5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534,5,229,529, and EP 590267), or obtained from a variety of commercialsources, including for example, Sigma Chemical Co., St. Louis, Mo.(T7402—from Taxus brevifolia).

Suramin is a polysulfonated naphthylurea compound that is typically usedas a trypanocidal agent. Briefly, Suramin blocks the specific cellsurface binding of various growth factors such as platelet derivedgrowth factor (“PDGF”), epidermal growth factor (“EGF”), transforminggrowth factor (“TGF-β”), insulin-like growth factor (“IGF-1”), andfibroblast growth factor (“βFGF”). Suramin may be prepared in accordancewith known techniques, or readily obtained from a variety of commercialsources, including for example Mobay Chemical Co., New York. (seeGagliardi et al., Cancer Res. 52:5073-5075, 1992; and Coffey, Jr., etal., J. of Cell. Phys. 132:143-148, 1987).

Tissue Inhibitor of Metalloproteinases-1 (“TIMP”) is secreted byendothelial cells which also secrete MTPases. TIMP is glycosylated andhas a molecular weight of 28.5 kDa. TIMP-1 regulates angiogenesis bybinding to activated metalloproteinases, thereby suppressing theinvasion of blood vessels into the extracellular matrix. TissueInhibitor of Metalloproteinases-2 (“TIMP-2”) may also be utilized toinhibit angiogenesis. Briefly, TIMP-2 is a 21 kDa nonglycosylatedprotein which binds to metalloproteinases in both the active and latent,pr benzyme forms. Both TIMP-1 and TIMP-2 may be obtained from commercialsources such as Synergen, Boulder, Colo.

Plasminogen Activator Inhibitor-1 (PA) is a 50 kDa glycoprotein which ispresent in blood platelets, and can also be synthesized by endothelialcells and muscle cells. PAI-1 inhibits t-PA and urokinase plasminogenactivator at the basolateral site of the endothelium, and additionallyregulates the fibrinolysis process. Plasminogen Activator Inhibitor-2(PAI-2) is generally found only in the blood under certain circumstancessuch as in pregnancy, and in the presence of tumors. Briefly, PAI-2 is a56 kDa protein which is secreted by monocytes and macrophages. It isbelieved to regulate fibrinolytic activity, and in particular inhibitsurokinase plasminogen activator and tissue plasminogen activator,thereby preventing fibrinolysis.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate (i.e., VO₃ ⁻) and orthovanadate (i.e.,VO₄ ³⁻) complexes such as, for example, ammonium metavanadate (i.e.,NH₄VO₃), sodium metavanadate (i.e., NaVO₃), and sodium orthovanadate(i.e., Na₃VO₄). Suitable vanadyl (i.e., VO²⁺) complexes include, forexample, vanadyl acetylacetonate and vanadyl sulfate including vanadylsulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate (i.e., WO₄ ²⁻)complexes include ammonium tungstate (i.e., (NH₄)₂WO₄), calciumtungstate (i.e., CaWO₄), sodium tungstate dihydrate (i.e., Na₂WO₄·2H₂O),and tungstic acid (i.e., H₂WO₄). Suitable tungsten oxides includetungsten (IV) oxide (i.e., WO₂) and tungsten (VI) oxide (i.e., WO₃).Suitable oxo molybdenum complexes include molybdate, molybdenum oxide,and molybdenyl complexes. Suitable molybdate (i.e., MoO₄ ²⁻) complexesinclude ammonium molybdate (i.e., (NH₄)₂MoO₄) and its hydrates, sodiummolybdate (i.e., Na₂MoO₄) and its hydrates, and potassium molybdate(i.e., K₂MoO₄) and its hydrates. Suitable molybdenum oxides includemolybdenum (VI) oxide (i.e., MoO₂), molybdenum (V I) oxide (i.e., MoO₃),and molybdic acid. Suitable molybdenyl (i.e., MoO₂ ²⁺) complexesinclude, for example, molybdenyl acetylacetonate. Other suitabletungsten and molybdenum complexes include hydroxo derivatives derivedfrom, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude Platelet Factor 4 (Sigma Chemical Co., #F1385); ProtamineSulphate (Clupeine) (Sigma Chemical Co., #P4505); Sulphated ChitinDerivatives (prepared from queen crab shells), (Sigma Chemical Co.,#C3641; Murata et al., Cancer Res. 51:22-26, 1991);. SulphatedPolysaccharide Peptidoglycan Complex (SP-PG) (the function of thiscompound may be enhanced by the presence of steroids such as estrogen,and tamoxifen citrate); Staurosporine (Sigma Chemical Co., #S4400);Modulators of Matrix Metabolism, including for example, proline analogs{[(L-azetidine-2-carboxylic acid (LACA) (Sigma Chemical Co., #A0760)),cishydroxyproline, d,L-3,4-dehydroproline (Sigma Chemical Co., #D0265),Thiaproline (Sigma Chemical Co., #T0631)], α,α-dipyridyl (Sigma ChemicalCo., #D7505), β-aminopropionitrile fumarate (Sigma Chemical Co., #A3134)J}; MDL 27032 (4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Merion MerrelDow Research Institute); Methotrexate (Sigma Chemical Co., #A6770;Hirata et al., Arthritis and Rheumatism 32:1065-1073, 1989);Mitoxantrone (Polverini and Novak, Biochem. Biophys. Res. Comm.140:901-907); Heparin (Folkman, Bio. Phar. 34:905-909, 1985; SigmaChemical Co., #P8754); Interferons (e.g., Sigma Chemical Co., #13265); 2Macroglobulin-serum (Sigma Chemical Co., #M7151); ChIMP-3 (Pavloff etal., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Sigma ChemicalCo., #C7268; Tomkinson et al., Biochem J. 286:475-480, 1992);β-Cyclodextrin Tetradecasulfate (Sigma Chemical Co., #C4767);Eponemycin; Camptothecin; Fumagillin (Sigma Chemical Co., #F6771;Canadian Patent No. 2,024,306; Ingber et al., Nature 348:555-557, 1990);Gold Sodium Thiomalate (“GST”; Sigma:G4022; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); (D-Penicillamine (“CDPT”; Sigma ChemicalCo., #P4875 or P5000(HCl)); β-1-anticollagenase-serum; α2-antiplasmin(Sigma Chem. Co.:A0914; Holmes et al., J. Biol. Chem. 262(4):1659-1664,1987); Bisantrene (National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide;Angostatic steroid; AGM-1470; carboxynaminolmidazole; metalloproteinaseinhibitors such as BB94 and the peptide CDPGYIGSR-NH₂ (SEQUENCE IDNO. 1) (Iwaki Glass, Tokyo, Japan).

Although the above anti-angiogenic factors have been provided for thepurposes of illustration, it should be understood that the presentinvention is not so limited. In particular, although certainanti-angiogenic factors are specifically referred to above, the presentinvention should be understood to include analogues, derivatives andconjugates of such anti-angiogenic factors. For example, paclitaxelshould be understood to refer to not only the common chemicallyavailable form of paclitaxel, but analogues (e.g., taxotere, as notedabove) and paclitaxel conjugates (e.g., paclitaxel-PEG,paclitaxel-dextran, or paclitaxel-xylos).

Anti-angiogenic compositions of the present invention may additionallycomprise a wide variety of compounds in addition to the anti-angiogenicfactor and polymeric carrier. For example, anti-angiogenic compositionsof the present invention may also, within certain embodiments of theinvention, also comprise one or more antibiotics, anti-inflammatories,anti-viral agents, anti-fungal agents and/or anti-protozoal agents.Representative examples of antibiotics included within the compositionsdescribed herein include: penicillins; cephalosporins such ascefadroxil, cefazolin, cefaclor; aminoglycosides such as gentamycin andtobramycin; sulfonamides such as sulfamethoxazole; and metronidazole.Representative examples of anti-inflammatories include: steroids such asprednisone, prednisolone, hydrocortisone, adrenocorticotropic hormone,and sulfasalazine; and non-steroidal anti-inflammatory drugs (“NSAIDS”)such as aspirin, ibuprofen, naproxen, fenoprofen, indomethacin, andphenylbutazone. Representative examples of antiviral agents includeacyclovir, ganciclovir, zidovudine. Representative examples ofantifungal agents include: nystatin, ketoconazole, griseofulvin,flucytosine, miconazole, clotrimazole. Representative examples ofantiprotozoal agents include: pentamidine isethionate, quinine,chloroquine, and mefloquine.

Anti-angiogenic compositions of the present invention may also containone or more hormones such as thyroid hormone, estrogen, progesterone,cortisone and/or growth hormone, other biologically active moleculessuch as insulin, as well as T_(H)1 (e.g., Interleukins -2, -12, and -15,gamma -interferon) or T_(H)2 (e.g., Interleukins -4 and -10) cytokines.

Within certain preferred embodiments of the invention, anti-angiogeniccompositions are provided which contain one or more compounds whichdisrupt microtubule function. Representative examples of such compoundsinclude paclitaxel (discussed above), estramustine (available fromSigma; Wang and Steams Cancer Res. 48:6262-6271, 1988), epothilone,curacin-A, colchicine, methotrexate, vinblastine and4-tert-butyl-[3-(2-chloroethyl) ureido] benzene (“tBCEU”).

Anti-angiogenic compositions of the present invention may also contain awide variety of other compounds, including for example: α-adrenergicblocking agents, angiotensin II receptor antagonists and receptorantagonists for histamine, serotonin, endothelin; inhibitors of thesodium/hydrogen antiporter (e.g. amiloride and its derivatives); agentsthat modulate intracellular Ca²⁺ transport such as L-type (e.g.,diltiazem, nifedipine, verapamil) or T-type Ca²⁺ channel blockers (e.g.,amiloride), calmodulin antagonists (e.g., H₇) and inhibitors of thesodium/calcium antiporter (e.g., amiloride); ap-1 inhibitors (fortyrosine kinases, protein kinase C, myosin light chain kinase,Ca²⁺/calmodulin kinase II, casein kinase II); anti-depressants (e.g.amytriptyline, fluoxetine, LUVOX® and PAXIL®); cytokine and/or growthfactors, as well as their respective receptors, (e.g., the interleukins,α, β or γ-IFN, GM-CSF, G-CSF, epidermal growth factor, transforminggrowth factors alpha and beta, TNF, and antagonists of vascularepithelial growth factor, endothelial growth factor, acidic or basicfibroblast growth factors, and platelet dervived growth factor);inhibitors of the IP₃ receptor (e.g., heparin); protease and collagenaseinhibitors (e.g., TIMPs, discussed above); nitrovasodilators (e.g.,isosorbide dinitrate); anti-mitotic agents (e.g., colchicine,anthracyclines and other antibiotics, folate antagonists and otheranti-metabolites, vinca alkaloids, nitrosoureas, DNA alkylating agents,topoisomerase inhibitors, purine antagonists and analogs, pyrimidineantagonists and analogs, alkyl sulfonates); immunosuppressive agents(e.g., adrenocorticosteroids, cyclosporine); sense or antisenseoligonucleotides (e.g., DNA, RNA, nucleic acid analogues (e.g., peptidenucleic acids) or any combinations of these); and inhibitors oftranscription factor activity (e.g., lighter d group transition metals).

Anti-angiogenic compositions of the present invention may also compriseadditional ingredients such as surfactants (either hydrophilic orhydrophobic; see Example 13), anti-neoplastic or chemotherapeutic agents(e.g., 5-fluorouracil, vincristine, vinblastine, cisplatin, doxyrubicin,adriamycin, or tamocifen), radioactive agents (e.g., Cu-64, Ga-67,Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125,I-131, Re-186, Re-188, Au-198, Au-199, Pb-203, At-211, Pb-212 andBi-212) or toxins (e.g., ricin, abrin, diphtheria toxin, cholera toxin,gelonin, pokeweed antiviral protein, tritin, Shigella toxin, andPseudomonas exotoxin A).

As noted above, anti-angiogenic compositions of the present inventioncomprise an anti-angiogenic factor and a polymeric carrier. In additionto the wide array of anti-angiogenic factors and other compoundsdiscussed above, anti-angiogenic compositions of the present inventionare provided in a wide variety of polymeric carriers, including forexample both biodegradable and non-biodegradable compositions.Representative examples of biodegradable compositions include albumin,gelatin, starch, cellulose, dextrans, polysaccharides, fibrinogen, poly(D,L lactide), poly (D,L-lactide-co-glycolide), poly (glycolide), poly(hydroxybutyrate), poly (alkylcarbonate) and poly (orthoesters) (seegenerally, Illum, L., Davids, S. S. (eds.) “Polymers in controlled DrugDelivery” Wright, Bristol, 1987; Arshady, J. Controlled Release 17:1-22,1991; Pitt, Int. J Phar. 59:173-196, 1990; Holland et al., J. ControlledRelease 4:155-0180, 1986). Representative examples of nondegradablepolymers include EVA copolymers, silicone rubber and poly(methylmethacrylate). Particularly preferred polymeric carriers includepoly (ethylene-vinyl acetate)(40% cross-linked), poly (D,L-lactic acid)oligomers and polymers, poly (L-lactic acid) oligomers and polymers,poly (glycolic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone), poly (valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly (lactic acid) with polyethylene glycol and blendsthereof.

Polymeric carriers may be fashioned in a variety of forms, including forexample, rod-shaped devices, pellets, slabs, or capsules (see, e.g.,Goodell et al., Am. J. Hosp. Pharm. 43:1454-1461, 1986; Langer et al.,“Controlled release of macromolecules from polymers”, in Biomedicalpolymers, Polymeric materials and pharmaceuticals for biomedical use,Goldberg, E. P., Nakagim, A. (eds.) Academic Press, pp. 113-137, 1980;Rhine et al., J. Pharm. Sci. 69:265-270, 1980; Brown et al., J. Pharm.Sci. 72:1181-1185, 1983; and Bawa et al., J. Controlled Release1:259-267, 1985). Anti-angiogenic factors may be linked by occlusion inthe matrices of the polymer, bound by covalent linkages, or encapsulatedin microcapsules. Within certain preferred embodiments of the invention,anti-angiogenic compositions are provided in non-capsular formulationssuch as microspheres (ranging from nanometers to micrometers in size),pastes, threads of various size, films and sprays.

Preferably, anti-angiogenic compositions of the present invention (whichcomprise one or more anti-angiogenic factors, and a polymeric carrier)are fashioned in a manner appropriate to the intended use. Withincertain aspects of the present invention, the anti-angiogeniccomposition should be biocompatible, and release one or moreanti-angiogenic factors over a period of several days to months. Forexample, “quick release” or “burst” anti-angiogenic compositions areprovided that release greater than 10%, 20%, or 25% (w/v) of ananti-angiogenic factor (e.g., paclitaxel) over a period of 7 to 10 days.Such “quick release” compositions should, within certain embodiments, becapable of releasing chemotherapeutic levels (where applicable) of adesired anti-angiogenic factor. Within other embodiments, “low release”anti-angiogenic compositions are provided that release less than 1%(w/v) of an anti-angiogenic factor over a period of 7 to 10 days.Further, anti-angiogenic compositions of the present invention shouldpreferably be stable for several months and capable of being producedand maintained under sterile conditions.

Within certain aspects of the present invention, anti-angiogeniccompositions may be fashioned in any size ranging from 50 nm to 500 μm,depending upon the particular use. For example, when used for thepurpose of tumor embolization (as discussed below), it is generallypreferable to fashion the anti-angiogenic composition in microspheres ofbetween 15 and 500 μm, preferably between 15 and 200 μm, and mostpreferably, between 25 and 150 μm. Alternatively, such compositions mayalso be readily applied as a “spray”, which solidifies into a film orcoating. Such sprays may be prepared from microspheres of a wide arrayof sizes, including for example, from 0.1 μm to 3 μm, from 10 μm to 30μm, and from 30 μm to 100 μm (see Example 8).

Anti-angiogenic compositions may also be prepared, given the disclosureprovided herein, for a variety of other applications. For example, foradministration to the cornea, the anti-angiogenic factors of the presentinvention may be incorporated into muco-adhesive polymers (e.g.,polyacrylic acids such as (CARBOPOL®, dextron, polymethacrylate, orstarch (see LeYung and Robinson, J. of Controlled Rel. 5:223, 1988)), ornanometer-sized microspheres (see generally, Kreuter J. ControlledRelease 16:169-176, 1991; Couvreur and Vauthier, J. Controlled Release17:187-198, 1991).

Anti-angiogenic compositions of the present invention may also beprepared in a variety of “paste” or gel forms. For example, within oneembodiment of the invention, anti-angiogenic compositions are providedwhich are liquid at one temperature (e.g., temperature greater than 37°C., such as 40° C., 45° C., 50° C., 55° C. or 60° C.), and solid orsemi-solid at another temperature (e.g., ambient body temperature, orany temperature lower than 37° C). Such “thermopastes” may be readilymade given the disclosure provided herein (see, e.g., Examples 10 and14).

Within yet other aspects of the invention, the anti-angiogeniccompositions of the present invention may be formed as a film.Preferably, such films are generally less than 5, 4, 3, 2, or 1, mmthick, more preferably less than 0.75 mm or 0.5 mm thick, and mostpreferably less than 500 μm to 100 μm thick. Such films are preferablyflexible with a good tensile strength (e.g., greater: than 50,preferably greater than 100, and more preferably greater than 150 or 200N/cm²), good adhesive properties (i.e., readily adheres to moist or wetsurfaces), and has controlled permeability. Representative examples ofsuch films are set forth below in the Examples (see e.g., Example 13).

Representative examples of the incorporation of anti-angiogenic factorssuch as those described above into a polymeric carriers is described inmore detail below in Examples 3, 4 and 8-15.

Polymeric Carriers for the Release of Hydrophobic Compounds

Within further aspects of the present invention, polymeric carriers areprovided which are adapted to contain and release a hydrophobiccompound, the carrier containing the hydrophobic compound in combinationwith a carbohydrate, protein or polypeptide. Within certain embodiments,the polymeric carrier contains or comprises regions, pockets, orgranules of one or more hydrophobic compounds. For example, within oneembodiment of the invention, hydrophobic compounds may be incorporatedwithin a matrix which contains the hydrophobic compound, followed byincorporation of the matrix within the polymeric carrier. A variety ofmatrices can be utilized in this regard, including for example,carbohydrates and polysaccharides such as starch, cellulose, dextran,methylcellulose, and hyaluronic acid, proteins or polypeptides such asalbumin, collagen and gelatin (see e.g., Example 31). Within alternativeembodiments, hydrophobic compounds may be contained within a hydrophobiccore, and this core contained within a hydrophilic shell. For example,as described in Example 38, paclitaxel may be incorporated into ahydrophobic core (e.g., of the poly D,L lactic acid-PEG or MePEGaggregate) which has a hydrophilic shell.

A wide variety of hydrophobic compounds may be released from thepolymeric carriers described above, including for example: certainhydrophobic compounds which disrupt microtubule function such aspaclitaxel and estramustine; hydrophobic proteins such as myelin basicprotein, proteolipid proteins of CNS myelin, hydrophobic cell wallprotein, porins, membrane proteins (EMBO J. 12(9):3409-3415, 1993),myelin oligodendrocyte glycoprotein (“MOG”) (Biochem. and Mol. Biol.Int. 30(5):945-958, 1993, P27 Cancer Res. 53(17):4096-4101, 1913,bacterioopsin, human surfactant protein (“HSB”; J. Biol. Chem.268(15):11160-11166, 1993), and SP-B or SP-C (Biochimica et BiophysicaActa 1105(1):161-169, 1992).

Arterial Embolization

In addition to the compositions described above, the present inventionalso provides a variety of methods which utilize the above-describedanti-angiogenic compositions. In particular, within one aspect of thepresent invention methods are provided for embolizing a blood vessel,comprising the step of delivering into the vessel a therapeuticallyeffective amount of an anti-angiogenic composition (as described above),such that the blood vessel is effectively occluded. Therapeuticallyeffective amounts suitable for occluding blood vessels may be readilydetermined given the disclosure provided below, and as described inExample 6. Within a particularly preferred embodiment, theanti-angiogenic composition is delivered to a blood vessel whichnourishes a tumor (see FIG. 13).

Briefly, there are a number of clinical situations (e.g., bleeding,tumor development) where it is desirable to reduce or abolish the bloodsupply to an organ or region. As described in greater detail below, thismay be accomplished by injecting anti-angiogenic compositions of thepresent invention into a desired blood vessel through a selectivelypositioned catheter (see FIG. 13). The composition travels via the bloodstream until it becomes wedged in the vasculature, thereby physically(or chemically) occluding the blood vessel. The reduced or abolishedblood flow to the selected area results in infarction (cell death due toan inadequate supply of oxygen and nutrients) or reduced blood loss froma damaged vessel.

For use in embolization therapy, anti-angiogenic compositions of thepresent invention are preferably non-toxic, thrombogenic, easy to injectdown vascular catheters, radio-opaque, rapid and permanent in effect,sterile, and readily available in different shapes or sizes at the timeof the procedure. In addition, the compositions preferably result in theslow (ideally, over a period of several weeks to months) release of ananti-angiogenic factor. Particularly preferred anti-angiogeniccompositions should have a predictable size of 15-200 μm after beinginjected into the vascular system. Preferably, they should not clumpinto larger particles either in solution or once injected. In addition,preferable compositions should not change shape or physical propertiesduring storage prior to use.

Embolization therapy may be utilized in at least three principal ways toassist in the management of neoplasms: (1) definitive treatment oftumors (usually benign); (2) for preoperative embolization; and (3) forpalliative embolization. Briefly, benign tumors may sometimes besuccessfully treated by embolization therapy alone. Examples of suchtumors include simple tumors of vascular origin (e.g., haemangiomas),endocrine tumors such as parathyroid adenomas, and benign bone tumors.

For other tumors, (e.g., renal adenocarcinoma), preoperativeembolization may be employed hours or days before surgical resection inorder to reduce operative blood loss, shorten the duration of theoperation, and reduce the risk of dissemination of viable malignantcells by surgical manipulation of the tumor. Many tumors may besuccessfully embolized preoperatively, including for examplenasopharyngeal tumors, glomus jugular tumors, meningiomas,chemodectomas, and vagal neuromas.

Embolization may also be utilized as a primary mode of treatment forinoperable malignancies, in order to extend the survival time ofpatients with advanced disease. Embolization may produce a markedimprovement in the quality of life of patients with malignant tumors byalleviating unpleasant symptoms such as bleeding, venous obstruction andtracheal compression. The greatest benefit from palliative tumorembolization, however, may be seen in patients suffering from thehumoral effects of malignant endocrine tumors, wherein metastases fromcarcinoid tumors and other endocrine neoplasms such as insulinomas andglucagonomas may be slow growing, and yet cause great distress by virtueof the endocrine syndromes which they produce.

In general, embolization therapy utilizing anti-angiogenic compositionsof the present invention is typically performed in a similar mariner,regardless of the site. Briefly, angiography (a road map of the bloodvessels) of the area to be embolized is first performed by injectingradiopaque contrast through a catheter inserted into an artery or vein(depending on the site to be embolized) as an X-ray is taken. Thecatheter may be inserted either percutaneously or by surgery. The bloodvessel is then embolized by refluxing anti-angiogenic compositions ofthe present invention through the catheter, until flow is observed tocease. Occlusion may be confirmed by repeating the angiogram.

Embolization therapy generally results in the distribution ofcompositions containing anti-angiogenic factors throughout theinterstices of the tumor or vascular mass to be treated. The physicalbulk of the embolic particles clogging the arterial lumen results in theocclusion of the blood supply. In addition to this effect, the presenceof an anti-angiogenic factor(s) prevents the formation of new bloodvessels to supply the tumor or vascular mass, enhancing the devitalizingeffect of cutting off the blood supply.

Therefore, it should be evident that a wide variety of tumors may beembolized utilizing the compositions of the present invention. Briefly,tumors are typically divided into two classes: benign and malignant. Ina benign tumor the cells retain their differentiated features and do notdivide in a completely uncontrolled manner. In addition, the tumor islocalized and nonmetastatic. In a malignant tumor, the cells becomeundifferentiated, do not respond to the body's growth and hormonalsignals, and multiply in an uncontrolled manner; the tumor is invasiveand capable of spreading to distant sites (metastasizing).

Within one aspect of the present invention, metastases (secondarytumors) of the liver may be treated utilizing embolization therapy.Briefly, a catheter is inserted via the femoral or brachial artery andadvanced into the hepatic artery by steering it through the arterialsystem under fluoroscopic guidance. The catheter is. advanced into thehepatic arterial tree as far as necessary to allow complete blockage ofthe blood vessels supplying the tumor(s), while sparing as many of thearterial branches supplying normal structures as possible. Ideally thiswill be a segmental branch of the hepatic artery, but it could be thatthe entire hepatic artery distal to the origin of the gastroduodenalartery, or even multiple separate arteries, will need to be blockeddepending on the extent of tumor and its individual blood supply. Oncethe desired catheter position is achieved, the artery is embolized byinjecting anti-angiogenic compositions (as described above) through thearterial catheter until flow in the artery to be blocked ceases,preferably even after observation for 5 minutes. Occlusion of the arterymay be confirmed by injecting radiopaque contrast through the catheterand demonstrating by fluoroscopy or X-ray film that the vessel whichpreviously filled with contrast no longer does so. The same proceduremay be repeated with each feeding artery to be occluded.

As noted above, both benign and malignant tumors may be embolizedutilizing compositions of the present invention. Representative examplesof benign hepatic tumors include Hepatocellular Adenoma, CavernousHaemangioma, and Focal Nodular Hyperplasia. Other benign tumors, whichare more rare and often do not have clinical manifestations, may also betreated. These include Bile Duct Adenomas, Bile Duct Cystadenomas,Fibromas, Lipomas, Leiomyomas, Mesotheliomas, Teratomas, Myxomas, andNodular Regenerative Hyperplasia.

Malignant Hepatic Tumors are generally subdivided into two categories:primary and secondary. Primary tumors arise directly from the tissue inwhich they are found. Thus, a primary liver tumor is derived originallyfrom the cells which make up the liver tissue (such as hepatocytes andbiliary cells). Representative examples of primary hepatic malignancieswhich may be treated by arterial embolization includeHepatocellularcarcinoma, Cholangiocarcinoma, Angiosarcoma,Cystadenocarcinoma, Squamous Cell Carcinoma, and Hepatoblastoma.

A secondary tumor, or metastasis, is a tumor which originated elsewherein the body but has now spread to a distant organ. The common routes formetastasis are direct growth into adjacent structures, spread throughthe vascular or lymphatic systems, and tracking along tissue planes andbody spaces (peritoneal fluid, cerebrospinal fluid, etc.). Secondaryhepatic tumors are one of the most common causes of death in the cancerpatient and are by far and away the most common form of liver tumor.Although virtually any malignancy can metastasize to the liver, tumorswhich are most likely to spread to the liver include: cancer of thestomach, colon, and pancreas; melanoma; tumors of the lung, oropharynx,and bladder; Hodgkin's and non-Hodgkin's lymphoma; tumors of the breast,ovary, and prostate. Each one of the above-named primary tumors hasnumerous different tumor types which may be treated by arterialembolization (for example, there are over 32 different types of ovariancancer).

As noted above, embolization therapy utilizing anti-angiogeniccompositions of the present invention may also be applied to a varietyof other clinical situations where it is desired to occlude bloodvessels. Within one aspect of the present invention, arteriovenousmalformation may be treated by administration of one of theabove-described compositions. Briefly, arteriovenous malformations(vascular malformations) refers to a group of diseases wherein at leastone (and most typically, many) abnormal communications between arteriesand veins occur, resulting in a local tumor-like mass composedpredominantly of blood vessels. Such disease may be either congenital oracquired.

Within one embodiment of the invention, an arteriovenous malformationmay be treated by inserting a catheter via the femoral or brachialartery, and advancing it into the feeding artery under fluoroscopicguidance. The catheter is preferably advanced as far as necessary toallow complete blockage of the blood vessels supplying the vascularmalformation, while sparing as many of the arterial branches supplyingnormal structures as possible (ideally this will be a single artery, butmost often multiple separate arteries may need to be occluded, dependingon the extent of the vascular malformation and its individual bloodsupply). Once the desired catheter position is achieved, each artery maybe embolized utilizing anti-angiogenic compositions of the presentinvention.

Within another aspect of the invention, embolization may be accomplishedin order to treat conditions of excessive bleeding. For example,menorrhagia (excessive bleeding with menstruation) may be readilytreated by embolization of uterine arteries. Briefly, the uterinearteries are branches of the internal iliac arteries bilaterally. Withinone embodiment of the invention, a catheter may be inserted via thefemoral or brachial artery, and advanced into each uterine artery bysteering it through the arterial system under fluoroscopic guidance. Thecatheter should be advanced as far as necessary to allow completeblockage of the blood vessels to the uterus, while sparing as manyarterial branches that arise from the uterine artery and supply normalstructures as possible. Ideally a single uterine artery on each side maybe embolized, but occasionally multiple separate arteries may need to beblocked depending on the individual blood supply. Once the desiredcatheter position is achieved, each artery may be embolized byadministration of the anti-angiogenic compositions as described above.

In a like manner, arterial embolization may be accomplished in a varietyof other conditions, including for example, for acute bleeding, vascularabnormalities, central nervous system disorders, and hypersplenism.

Use of Anti-Angiogenic Compositions as Coatings for Stents

As noted above, the present invention also provides stents, comprising agenerally tubular structure (which includes for example, spiral shapes),the surface of which is coated with a composition as described above.Briefly, a stent is a scaffolding, usually cylindrical in shape, thatmay be inserted into a body passageway (e.g., bile ducts) or a portionof a body passageway, which has been narrowed, irregularly contured,obstructed, or occluded by a disease process (e.g., ingrowth by a tumor)in order to prevent closure or reclosure of the passageway. Stents actby physically holding open the walls of the body passage into which theyare inserted.

A variety of stents may be utilized within the context of the presentinvention, including for example, esophageal stents, vascular stents,biliary stents, pancreatic stents, ureteric and urethral stents,lacrimal stents, Eustachian tube stents, fallopian tube stents andtracheal/bronchial stents.

Stents may be readily obtained from commercial sources, or constructedin accordance with well-known techniques. Representative examples ofstents include those described in U.S. Pat. No. 4,768,523, entitled“Hydrogel Adhesive;” U.S. Pat. No. 4,776,337, entitled “ExpandableIntraluminal Graft, and Method and Apparatus for Implanting andExpandable Intraluminal Graft;” U.S. Pat. No. 5,041,126 entitled“Endovascular Stent and Delivery System;” U.S. Pat. No. 5,052,998entitled “Indwelling Stent and Method of Use;” U.S. Pat. No. 5,064,435entitled “Self-Expanding Prosthesis Having Stable Axial Length;” U.S.Pat. No. 5,089,606, entitled “Water-insoluble Polysaccharide HydrogelFoam for Medical Applications;” U.S. Pat. No. 5,147,370, entitled“Nitinol Stent for Hollow Body Conduits;” U.S. Pat. No. 5,176,626,entitled “Indwelling Stent;” U.S. Pat. No. 5,213,580, entitled“Biodegradable polymeric Endoluminal Sealing Process;” and U.S. Pat. No.5,328,471, entitled “Method and Apparatus for Treatment of Focal Diseasein Hollow Tubular Organs and Other Tissue Lumens.”

Stents may be coated with anti-angiogenic compositions oranti-angiogenic factors of the present invention in a variety ofmanners, including for example: (a) by directly affixing to the stent ananti-angiogenic composition (e.g., by either spraying the stent with apolymer/drug film, or by dipping the stent into a polymer/drugsolution), (b) by coating the stent with a substance such as a hydrogelwhich will in turn absorb the anti-angiogenic composition (oranti-angiogenic factor above), (c) by interweaving anti-angiogeniccomposition coated thread (or the polymer itself formed into a thread)into the stent structure, (d) by inserting the stent into a sleeve ormesh which is comprised of or coated with an anti-angiogeniccomposition, or (e) constructing the stent itself with ananti-angiogenic composition. Within preferred embodiments of theinvention, the composition should firmly adhere to the stent duringstorage and at the time of insertion, and should not be dislodged fromthe stent when the diameter is expanded from its collapsed size to itsfull expansion size. The anti-angiogenic composition should alsopreferably not degrade during storage, prior to insertion, or whenwarmed to body temperature after expansion inside the body. In addition,it should preferably coat the stent smoothly and evenly, with a uniformdistribution of angiogenesis inhibitor, while not changing the stentcontour. Within preferred embodiments of the invention, theanti-angiogenic composition should provide a uniform, predictable,prolonged release of the anti-angiogenic factor into the tissuesurrounding the stent once it has been deployed. For vascular stents, inaddition to the above properties, the composition should not render thestent thrombogenic (causing blood clots to form), or cause significantturbulence in blood flow (more than the stent itself would be expectedto cause if it was uncoated).

Within another aspect of the present invention, methods are provided forexpanding the lumen of a body passageway, comprising inserting a stentinto the passageway, the stent having a generally tubular structure, thesurface of the structure being coated with an anti-angiogeniccomposition (or, an anti-angiogenic factor alone), such that thepassageway is expanded. A variety of embodiments are described belowwherein the lumen of a body passageway is expanded in order to eliminatea biliary, esophageal, tracheal/bronchial, urethral or vascularobstruction. In addition, a representative example is described in moredetail below in Example 7.

Generally, stents are inserted in a similar fashion regardless of thesite or the disease being treated. Briefly, a preinsertion examination,usually a diagnostic imaging procedure, endoscopy, or directvisualization at the time of surgery, is generally first performed inorder to determine the appropriate positioning for stent insertion. Aguidewire is then advanced through the lesion or proposed site ofinsertion, and over this is passed a delivery catheter which allows astent in its collapsed form to be inserted. Typically, stents arecapable of being compressed, so that they can be inserted through tinycavities via small catheters, and then expanded to a larger diameteronce they are at the desired location. Once expanded, the stentphysically forces the walls of the passageway apart and holds them open.As such, they are capable of insertion via a small opening, and yet arestill able to hold open a large diameter cavity or passageway. The stentmay be self-expanding (e.g., the Wallstent and Gianturco stents),balloon expandable (e.g., the Palmaz stent and Strecker stent), orimplanted by a change in temperature (e.g., the Nitinol stent).

Stents are typically maneuvered into place under radiologic or directvisual control, taking particular care to place the stent preciselyacross the narrowing in the organ being treated. The delivery catheteris then removed, leaving the stent standing on its own as a scaffold. Apost insertion examination, usually an x-ray, is often utilized toconfirm appropriate positioning.

Within a preferred embodiment of the invention, methods are provided foreliminating biliary obstructions, comprising inserting a biliary stentinto a biliary passageway, the stent having a generally tubularstructure, the surface of the structure being coated with a compositionas described above, such that the biliary obstruction is eliminated.Briefly, tumor overgrowth of the common bile duct results in progressivecholestatic jaundice which is incompatible with life. Generally, thebiliary system which drains bile from the liver into the duodenum ismost. often obstructed by (1) a tumor composed of bile duct cells(cholangiocarcinoma), (2) a tumor which invades the bile duct (e.g.,pancreatic carcinoma), or (3) a tumor which exerts extrinsic pressureand compresses the bile duct (e.g., enlarged lymph nodes).

Both primary biliary tumors, as well as other tumors which causecompression of the biliary tree may be treated utilizing the stentsdescribed herein. One example of primary biliary tumors areadenocarcinomas (which are also called Klatskin tumors when found at thebifurcation of the common hepatic duct). These tumors are also referredto as biliary carcinomas, choledocholangiocarcinomas, or adenocarcinomasof the biliary system. Benign tumors which affect the bile duct (e.g.,adenoma of the biliary system), and, in rare cases, squamous cellcarcinomas of the bile duct and adenocarcinomas of the gallbladder, mayalso cause compression of the biliary tree and therefore, result inbiliary obstruction.

Compression of the biliary tree is most commonly due to tumors of theliver and pancreas which compress and therefore obstruct the ducts. Mostof the tumors from the pancreas arise from cells of the pancreaticducts. This is a highly fatal form of cancer (5%. of all cancer deaths;26,000 new cases per year in the U.S.) with an average of 6 monthssurvival and a 1 year survival rate of only 10%. When these tumors arelocated in the head of the pancreas they frequently cause biliaryobstruction, and this detracts significantly from the quality of life ofthe patient. While all types of pancreatic tumors are generally referredto as “carcinoma of the pancreas” there are histologic subtypesincluding: adenocarcinoma, adenosquamous carcinoma, cystadeno-carcinoma,and acinar cell carcinoma. Hepatic tumors, as discussed above, may alsocause compression of the biliary tree, and therefore cause obstructionof the biliary ducts.

Within one embodiment of the invention, a biliary stent is firstinserted into a biliary passageway in one of several ways: from the topend by inserting a needle through the abdominal wall and through theliver (a percutaneous transhepatic cholangiogram or “PTC”); from thebottom end by cannulating the bile duct through an endoscope insertedthrough the mouth, stomach, and duodenum (an endoscopic retrogradecholangiogram or “ERCP”); or by direct incision during a surgicalprocedure. A preinsertion examination, PTC, ERCP, or directvisualization at the time of surgery should generally be performed todetermine the appropriate position for stent insertion. A guidewire isthen advanced through the lesion, and over this a delivery catheter ispassed to allow the stent to be inserted in its collapsed form. If thediagnostic exam was a PTC, the guidewire and delivery catheter isinserted via the abdominal wall, while if the original exam was an ERCPthe stent may be placed via the mouth. The stent is then positionedunder radiologic, endoscopic, or direct visual control taking particularcare to place it precisely across the narrowing in the bile duct. Thedelivery catheter is then removed leaving the stent standing as ascaffolding which holds the bile duct open. A further cholangiogram maybe performed to document that the stent is appropriately positioned.

Within yet another embodiment of the invention, methods are provided foreliminating esophageal obstructions, comprising inserting an esophagealstent into an esophagus, the stent having a generally tubular structure,the surface of the structure being coated with an anti-angiogeniccomposition as described above, such that the esophageal obstruction iseliminated. Briefly, the esophagus is the hollow tube which transportsfood and liquids from the mouth to the stomach. Cancer of the esophagusor invasion by cancer arising in adjacent organs (e.g., cancer of thestomach or lung) results in the inability to swallow food or saliva.Within this embodiment, a preinsertion examination, usually a bariumswallow or endoscopy should generally be performed in order to determinethe appropriate position for stent insertion. A catheter or endoscopemay then be positioned through the mouth, and a guidewire is advancedthrough the blockage. A stent delivery catheter is passed over theguidewire under radiologic or endoscopic control, and a stent is placedprecisely across the narrowing in the esophagus. A post insertionexamination, usually a barium swallow x-ray, may be utilized to confirmappropriate positioning.

Within other embodiments of the invention, methods are provided foreliminating tracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi, the stent having agenerally tubular structure, the surface of which is coated with ananti-angiogenic composition as described above, such that thetracheal/bronchial obstruction is eliminated. Briefly, the trachea andbronchi are tubes which carry air from the mouth and nose to the lungs.Blockage of the trachea by cancer, invasion by cancer arising inadjacent organs (e.g., cancer of the lung), or collapse of the tracheaor bronchi due to chondromalacia (weakening of the cartilage rings)results in inability to breathe. Within this embodiment of theinvention, preinsertion examination, usually an endoscopy, shouldgenerally be performed in order to determine the appropriate positionfor stent insertion. A catheter or endoscope is then positioned throughthe mouth, and a guidewire advanced through the blockage. A deliverycatheter is then passed over the guidewire in order to allow a collapsedstent to be inserted. The stent is placed under radiologic or endoscopiccontrol in order to place it precisely across the narrowing. Thedelivery catheter may then be removed leaving the stent standing as ascaffold on its own. A post insertion examination, usually abronchoscopy may be utilized to confirm appropriate positioning.

Within another embodiment of the invention, methods are provided foreliminating urethral obstructions, comprising inserting a urethral stentinto a urethra, the stent having a generally tubular structure, thesurface of the structure being coated with an anti-angiogeniccomposition as described above, such that the urethral obstruction iseliminated. Briefly, the urethra is the tube which drains the bladderthrough the penis. Extrinsic narrowing of the urethra as it passesthrough the prostate, due to hypertrophy of the prostate, occurs invirtually every man over the age of 60 and causes progressive difficultywith urination. Within this embodiment, a preinsertion examination,usually an endoscopy or urethrogram should generally first be performedin order to determine the appropriate position for stent insertion,which is above the external urinary sphincter at the lower end, andclose to flush with the bladder neck at the upper end. An endoscope orcatheter is then positioned through the penile opening and a guidewireadvanced into the bladder. A delivery catheter is then passed over theguidewire in order to allow stent insertion. The delivery catheter isthen removed, and the stent expanded into place. A post insertionexamination, usually endoscopy or retrograde urethrogram, may beutilized to confirm appropriate position.

Within another embodiment of the invention, methods are provided foreliminating vascular obstructions, comprising inserting a vascular stentinto a blood vessel, the stent having a generally tubular structure, thesurface of the structure being coated with an anti-angiogeniccomposition as described above, such that the vascular obstruction iseliminated. Briefly, stents may be placed in a wide array of bloodvessels, both arteries and veins, to prevent recurrent stenosis at thesite of failed angioplasties, to treat narrowings that would likely failif treated with angioplasty, and to treat post surgical narrowings(e.g., dialysis graft stenosis). Representative examples of suitablesites include the iliac, renal, and coronary arteries, the superior venacava, and in dialysis grafts. Within one embodiment, angiography isfirst performed in order to localize the site for placement of thestent. This is typically accomplished by injecting radiopaque contrastthrough a catheter inserted into an artery or vein as an x-ray is taken.A catheter may then be inserted either percutaneously or by surgery intothe femoral artery, brachial artery, femoral vein, or brachial vein, andadvanced into the appropriate blood vessel by steering it through thevascular system under fluoroscopic guidance. A stent may then bepositioned across the vascular stenosis. A post insertion angiogram mayalso be utilized in order to confirm appropriate positioning.

Use of Anti-Angiogenic Compositions in Surgical Procedures

As noted above, anti-angiogenic compositions may be utilized in a widevariety of surgical procedures. For example, within one aspect of thepresent invention an anti-angiogenic compositions (in the form of, forexample, a spray or film) may be utilized to coat or spray an area priorto removal of a tumor, in order to isolate normal surrounding tissuesfrom malignant tissue, and/or to prevent the spread of disease tosurrounding tissues. Within other aspects of the present invention,anti-angiogenic compositions (e.g., in the form of a spray) may bedelivered via endoscopic procedures in order to coat tumors, or inhibitangiogenesis in a desired locale. Within yet other aspects of thepresent invention, surgical meshes which have been coated withanti-angiogenic compositions of the present invention may be utilized inany procedure wherein a surgical mesh might be utilized. For example,within one embodiment of the invention a surgical mesh ladened with ananti-angiogenic composition may be utilized during abdominal cancerresection surgery (e.g., subsequent to colon resection) in order toprovide support to the structure, and to release an amount of theanti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering ananti-angiogenic composition as described above to the resection marginsof a tumor subsequent to excision, such that the local recurrence ofcancer and the formation of new blood vessels at the site is inhibited.Within one embodiment of the invention, the anti-angiogeniccomposition(s) (or anti-angiogenic factor(s) alone) are administereddirectly to the tumor excision site (e.g., applied by swabbing, brushingor otherwise coating the resection margins of the tumor with theanti-angiogenic composition(s) or factor(s)). Alternatively, theanti-angiogenic composition(s) or factor(s) may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compositionsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, anti-angiogenic compositions(as described above) may be administered to the resection margin of awide variety of tumors, including for example, breast, colon, brain andhepatic tumors. For example, within one embodiment of the invention,anti-angiogenic compositions may be administered to the site of aneurological tumor subsequent to excision, such that the formation ofnew blood vessels at the site are inhibited. Briefly, the brain ishighly functionally localized; i.e., each specific anatomical region isspecialized to carry out a specific function. Therefore it is thelocation of brain pathology that is often more important than the type.A relatively small lesion in a key area can be far more devastating thana much larger lesion in a less important area. Similarly, a lesion onthe surface of the brain may be easy to resect surgically, while thesame tumor located deep in the brain may not (one would have to cutthrough too many vital structures to reach it). Also, even benign tumorscan be dangerous for several reasons: they may grow in a key area andcause significant damage; even though they would be cured by surgicalresection this may not be possible; and finally, if left unchecked theycan cause increased intracranial pressure. The skull is an enclosedspace incapable of expansion. Therefore, if something is growing in onelocation, something else must be being compressed in anotherlocation—the result is increased pressure in the skull or increasedintracranial pressure. If such a condition is left untreated, vitalstructures can be compressed, resulting in death. The incidence of CNS(central nervous system) malignancies is 8-16 per 100,000. The prognosisof primary malignancy of the brain is dismal, with a median survival ofless than one year, even following surgical resection. These tumors,especially gliomas, are predominantly a local disease which recur within2 centimeters of the original focus of disease after surgical removal.

Representative examples of brain tumors which may be treated utilizingthe compositions and methods described herein include Glial Tumors (suchas Anaplastic Astrocytoma, Glioblastoma Multiform, PilocyticAstrocytoma, Oligodendroglioma, Ependymoma, Myxopapillary Ependymoma,Subependymoma, Choroid Plexus Papilloma); Neuron Tumors (e.g.,Neuroblastoma, Ganglioneuroblastoma, Ganglioneuroma, andMedulloblastoma); Pineal Gland Tumors (e.g., Pineoblastoma andPineocytoma); Menigeal Tumors (e.g., Meningioma, MeningealHemangiopericytoma, Meningeal Sarcoma); Tumors of Nerve Sheath Cells(e.g., Schwannoma (Neurolemmoma) and Neurofibroma); Lymphomas (e.g.,Hodgkin's and Non-Hodgkin's Lymphoma (including numerous subtypes, bothprimary and secondary); Malformative Tumors (e.g., Craniopharyngioma,Epidermoid Cysts, Dermoid Cysts and Colloid Cysts); and MetastaticTumors (which can be derived from virtually any tumor, the most commonbeing from lung, breast, melanoma, kidney, and gastrointestinal tracttumors).

Inflammatory Arthritis

Inflammatory arthritis is a serious health problems in developedcountries, particularly given the increasing number of aged individuals.For example, one form of inflammatory arthritis, rheumatoid arthritis(RA) is a multisystem chronic, relapsing, inflammatory disease ofunknown cause. Although many organs can be affected, RA is basically asevere form of chronic synovitis that sometimes leads to destruction andankylosis of affected joints (taken from Robbins Pathological Basis ofDisease, by R. S. Cotran, V. Kumar, and S. L. Robbins, W. B. SaundersCo., 1989). Pathologically the disease is characterized by a markedthickening of the synovial membrane which forms villous projections thatextend into the joint space, multilayering of the synoviocyte lining(synoviocyte proliferation), infiltration of the synovial membrane withwhite blood cells (macrophages, lymphocytes, plasma cells, and lymphoidfollicles; called an “inflammatory synovitis”), and deposition of fibrinwith cellular necrosis within the synovium. The tissue formed as aresult of this process is called pannus and eventually the pannus growsto fill the joint space. The pannus develops an extensive network of newblood vessels through the process of angiogenesis which is essential tothe evolution of the synovitis. Release of digestive enzymes [matrixmetalloproteinases (e.g., collagenase, stromelysin)] and other mediatorsof the inflammatory process (e.g., hydrogen peroxide, superoxides,lysosomal enzymes, and products of arachadonic acid metabolism) from thecells of the pannus tissue leads to the progressive destruction of thecartilage tissue. The pannus invades the articular cartilage leading toerosions and fragmentation of the cartilage tissue. Eventually there iserosion of the subchondral bone with fibrous ankylosis and ultimatelybony ankylosis, of the involved joint.

It is generally believed, but not conclusively proven, that RA is anautoimmune disease, and that many different arthriogenic stimuliactivate the immune response in the immunogenetically susceptible host.Both exogenous infectious agents (Ebstein-Barr Virus, Rubella virus,Cytomegalovirus, Herpes Virus, Human T-cell Lymphotropic Virus,Mycoplasma, and others) and endogenous proteins (collagen,proteoglycans, altered immunoglobulins) have been implicated as thecausative agent which triggers an inappropriate host immune response.Regardless of the inciting agent, autoimmunity plays a role in theprogression of the disease. In particular, the relevant antigen isingested by antigen-presenting cells (macrophages or dendritic cells inthe synovial membrane), processed, and presented to T lymphocytes. The Tcells initiate a cellular immune response and stimulate theproliferation and differentiation of B lymphocytes into plasma cells.The end result is the production of an excessive inappropriate immuneresponse directed against the host tissues [e.g., antibodies directedagainst Type II collagen, antibodies directed against the Fc portion ofautologous IgG (called “Rheumatoid Factor”)]. This further amplifies theimmune response and hastens the destruction of the cartilage tissue.Once this cascade is initiated numerous mediators of cartilagedestruction are responsible for the progression of rheumatoid arthritis.

Thus, within one aspect of the present invention, methods are providedfor treating or preventing inflammatory arthritis (e.g., rheumatoidarthritis) comprising the step of administering to a patient atherapeutically effective amount of an anti-angiogenic factor oranti-angiogenic composition to a joint. Within a preferred embodiment ofthe invention, anti-angiogenic factors (including anti-angiogeniccompositions, as described above) may be administered directly byintra-articular injection, as a surgical paste, or as an oral agent(e.g., containing the anti-angiogenic factor thalidomide). Onerepresentative example of such a method is set forth in more detailbelow in Example 19.

As utilized within the context of the present invention, it should beunderstood that efficatious administration of the anti-angiogenicfactors and compositions described herein may be assessed in severalways, including: (1) by preventing or lessening the pathological and/orclinical symptoms associated with rheumatoid arthritis; (2) bydownregulating the white blood cell response which initiates theinflammatory cascade and results in synovitis, swelling, pain, andtissue destruction; (3) by inhibiting the “tumor-like” proliferation ofsynoviocytes that leads to the development of a locally invasive anddestructive pannus tissue; (4) by decreasing the production/activity ofmatrix metalloproteinases produced by white blood cells, synoviocytes,chondrocytes, and endothelial cells, which degrade the cartilage matrixand result in irreversible destruction of the articular cartilage; and(5) by inhibiting blood vessel formation which provides the frameworkand nutrients necessary for the growth and development of the pannustissue. Furthermore, the anti-angiogenic factors or compositions shouldnot be toxic to normal chondrocytes at therapeutic levels. Each of theseaspects will be discussed in more detail below.

A. Inflammatory Response

Neutrophils are found in abundance in the synovial fluid, but only insmall numbers in the synovial membrane itself. It is estimated that morethan 1 billion neutrophils enter a moderately inflamed rheumatoid kneejoint each day (Hollingsworth et al., 1967) and remain there because nopathway exists by which they can leave the joint. These cells releasereactive free radicals and lysosomal enzymes which degrade the cartilagetissue. Other PMN products such as prostaglandins and leukotrienesaugment the inflammatory response and recruit more inflammatory cellsinto the joint tissue.

Lymphocytes, particularly T cells, are present in abundance in thediseased synovial tissue. Activated T cells produce a variety oflymphokines and cooperate with B cells to produce autoantibodies. Tcells products result in the activation macrophages, a cell which isthought to have an important role in the pathology of the disease. Themacrophages produce a variety destructive lysosomal enzymes,prostaglandins, and monokines and are also capable of stimulatingangiogenesis. One of the more important monokines secreted bymacrophages is IL-1. Briefly, IL-1 is known to: stimulate synthesis andrelease of collagenase by synoviocytes and synovial fibroblasts, inhibitproteoglycan synthesis by chondrocytes, activate osteoclasts, inducechanges in the endothelium of the synovial vasculature [stimulation ofendothelial production of plasminogen activator and colony stimulatingfactor, expression of leukocyte adhesion molecules, promotion ofprocoagulant activity (Wider et al., 1991)], and act as achemoattractant for lymphocytes and neutrophils.

Within one embodiment, downregulation of the white blood cell response,or inhibition of the inflammatory response, may be assessed bydetermination of the effect of the anti-angiogenic factor oranti-angiogenic composition on the response of neutrophils stimulatedwith opsonized CPPD crystals or opsonized zyrosan. Such methods areillustrated in more detail below in Example 22.

B. Synoviocyte Hyperplasia

During the development of RA, the synovial lining cells become activatedby products of inflammation or through phagocytosis of immune complexes.Several subtypes of synovial lining cells have been identified and allof them become intensely activated and undergo excessive hyperplasia andgrowth when stimulated. As the synovial tissue organizes to form apannus, the number of synoviocytes, blood vessels, connective tissueelements, and inflammatory cells increases to form a mass 100 times itsoriginal size. In many ways, the synovitis in rheumatoid arthritisbehaves much like a localized neoplasia (Harris, 1990). In fact,cultured rheumatoid synovial cells develop the phenotypiccharacteristics of anchorage-independent growth usually associated withneoplastic cells if they given sufficient plateletderived growth factor(Lafyatis et al., 1989). In addition, the synoviocytes also producelarge amounts of collagenase, stromelysin, prostaglandins, andInterleukin-1.

The tumor-like proliferation of the cells of the synovial connectivetissue stroma (synoviocytes, fibroblast-like cells and neovasculartissue) produces a pannus with many features of a localized malignancy.Supporting this tumor analogy are several findings: the pannus expresseshigh levels of oncoproteins such as c-myc and c-fos, producesmetalloproteinases to facilitate surrounding tissue invasion, expresscytoskeletal markers characteristic of poorly differentiated mesenchymaltissue (e.g., vimentin); synoviocytes in vitro grow rapidly, do notcontact inhibit, form foci, and can be grown under anchorage-independentconditions in soft agarose; and pannus tissue is capable of inducing thegrowth of a supporting vasculature (i.e. angiogenesis). All thesefindings are suggestive of a tissue in which normal growth regulation asbeen lost.

Within one embodiment, inhibition of synoviocyte proliferation may bedetermined by, for example, analysis of ³H-thymidine incorporation intosynoviocytes, or in vitro synoviocyte proliferation. Such methods areillustrated in more detail below in Example 23.

C. Matrix Metalloproteinases (MMP)

Irreparable degradation of the cartilage extracellular matrix isbelieved to be largely due to the enzymatic action of matrixmetalloproteinases on the components of the cartilage matrix. Althoughnumerous other enzymes are likely involved in the development of RA,collagenase (MMP-1) and stromelysin (MMP-3) play an important role(Vincetti et al., 1994) in disease progression. These enzymes arecapable of degrading type 11 collagen and proteoglycans respectively;the 2 major extracellular components of cartilage tissue. Cytokines suchas IL-1, epidermal growth factor (EGF), platelet-derived growth factor,and tumor necrosis factor are all potent stimulators of collagenase andstromelysin production. As described above, numerous cell types found inthe arthritic joint (white blood cells, synoviocytes, endothelial cells,and chondrocytes) are capable of synthesizing and secreting MMPS.

In proliferating rheumatoid synovial tissue, collagenase and stromelysinbecome the major gene products of the pannus and may comprise as much as2% of the messenger RNAs produced by the synovial fibroblasts(Brinkerhoff and Auble, 1990). Increased levels of collagenase andstromelysin are present in the cartilage of patients with RA and thelevel of enzyme activity in the joint correlates well with the severityof the lesion (Martel-Pelletier et al., 1993; Walakovitis et al., 1992).Because these enzymes are fundamental to the pathology of RA and resultin irreversible cartilage damage, many therapeutic strategies have beendevised to inhibit their effects.

Numerous naturally present inhibitors of MMP activity have beenidentified and named “TIMPS” for Tissue Inhibitors ofMetalloproteinases. Many of these protein inhibitors bind with theactive MMPs to form 1:1 noncovalent complexes which inactivate the MMPenzymes. The TIMPs are produced locally by chondrocytes and synovialfibroblasts and are likely responsible for the normal regulation ofconnective tissue degradation. It is thought that much of the damage tothe cartilage matrix is due to a local imbalance between MMP and TIMPactivity. This is probably due to increased production ofmetalloproteinases while the production of TIMPs remains at a normal orconstant level (Vincetti et al., 1994). To overcome this, therapeuticstrategies have been designed to add exogenous TIMPs (e.g., thechemically modified tetracycline molecules, collagen substrateanalogues) or to upregulate TIMP production (retinoids, transforminggrowth factor β, IL-6, IL-11, oncostatin M) in an effort to restore theenzymatic balance. However this approach has yet to translate intosignificant clinical results.

An alternative approach is to inhibit or downregulate the production ofthe MMPs to restore a normal balance of activity. Naturally occurringcompounds (TNFβ, all-trans retinoic acid) and synthetic compounds(retinoids, glucocorticoid hormones) have been demonstrated to inhibitMMP activity by suppressing transcription and synthesis of theseproteins. A post-transcriptional method of blocking MMP release couldalso be expected to result in a decrease in the amount of MMP producedand an improved balance between MMP and TIMP activity in the joint.

Within one embodiment, a decrease in the production or activity of MMP'smay be determined by, for example, analysis of IL-1 induced collagenaseexpression. One such method is illustrated in more detail below inExample 24.

D. Angiogenesis

The development of an extensive network of new blood vessels isessential to the development of the synovitis present in rheumatoidarthritis (Harris, 1990; Folkman et al., 1989; Sano et al., 1990).Several local mediators such as plateletderived growth factor (PDGF),TGF-β, and fibroblast growth factor (FGF) are likely responsible for theinduction and perpetuation of neovascularization within the synovium.Pannus tissue composed of new capillaries and synovial connective tissueinvades and destroys the articular cartilage. The migrating angiogenicvessels themselves produce and secrete increased levels ofmetalloproteinases such as collagenase and stromelysin capable ofdegrading the cartilage matrix (Case et al., 1989). The newly formedvessels are also quite “leaky” with gaps present between themicrovascular endothelial cells. This facilitates the exudation ofplasma proteins into the synovium (which increases swelling), enhancesWBCs movement from the circulation into the pannus tissue (whichincreases inflammation), and leads to the perivascular accumulation ofmononuclear inflammatory cells (Wilder et al., 1991).

In summary, the endothelial tissue plays an important role in thedevelopment of this disease by expressing the necessary surfacereceptors to allow inflammatory cells to leave the circulation and enterthe developing pannus, secreting proteolytic enzymes capable ofdegrading the cartilage matrix, and proliferating to form the newvessels (angiogenesis) required for the pannus tissue to increase insize and invade adjacent tissues.

Within one embodiment, inhibition of new blood vessel formation may bereadily determined in a variety of asays, including the CAM assaydescribed above and within Example 2.

Neovascular Diseases of the Eye

As noted above, the present invention also provides methods for treatingneovascular diseases of the eye, including for example, cornealneovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroblasia and macular degeneration.

Briefly, corneal neovascularization as a result of injury to theanterior segment is a significant cause of decreased visual acuity andblindness, and a major risk factor for rejection of corneal allografts.As described by Burger et al., Lab. Invest. 48:169-180, 1983, cornealangiogenesis involves three phases: a pre-vascular latent period, activeneovascularization, and vascular maturation and regression. The identityand mechanism of various angiogenic factors, including elements of theinflammatory response, such as leukocytes, platelets, cytokines, andeicosanoids, or unidentified plasma constituents have yet to berevealed.

Currently no clinically satisfactory therapy exists for inhibition ofcorneal neovascularization or regression of existing corneal newvessels. Topical corticosteroids appear to have some clinical utility,presumably by limiting stromal inflammation.

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of an anti-angiogenic composition (as described above)to the cornea, such that the formation of blood vessels is inhibited.Briefly, the cornea is a tissue which normally lacks blood vessels. Incertain pathological conditions however, capillaries may extend into thecornea from the pericorneal vascular plexus of the limbus. When thecornea becomes vascularized, it also becomes clouded, resulting in adecline in the patient's visual acuity. Visual loss may become completeif the cornea completely opacitates.

Blood vessels can enter the cornea in a variety of patterns and depths,depending upon the process which incites the neovascularization. Thesepatterns have been traditionally defined by ophthalmologists in thefollowing types: pannus trachomatosus, pannus leprosus, pannusphylctenulosus, pannus degenerativus, and glaucomatous pannus. Thecorneal stroma may also be invaded by branches of the anterior ciliaryartery (called interstitial vascularization) which causes severaldistinct clinical lesions: terminal loops, a “brush-like” pattern, anumbel form, a lattice form, interstitial arcades (from episcleralvessels), and aberrant irregular vessels.

A wide variety of disorders can result in corneal neovascularization,including for example, corneal infections (e.g., trachoma, herpessimplex keratitis, leishmaniasis and onchocerciasis), immunologicalprocesses (e.g., graft rejection and Stevens-Johnson's syndrome), alkaliburns, trauma, inflammation (of any cause), toxic and nutritionaldeficiency states, and as a complication of wearing contact lenses.

While the cause of corneal neovascularization may vary, the response ofthe cornea to the insult and the subsequent vascular ingrowth is similarregardless of the cause. Briefly, the location of the injury appears tobe of importance as only those lesions situated within a criticaldistance of the limbus will incite an angiogenic response. This islikely due to the fact that the angiogenic factors responsible foreliciting the vascular invasion are created at the site of the lesion,and must diffuse to the site of the nearest blood vessels (the limbus)in order to exert their effect. Past a certain distance from the limbus,this would no longer be possible and the limbic endothelium would not beinduced to grow into the cornea. Several angiogenic factors are likelyinvolved in this process, many of which are products of the inflammatoryresponse. Indeed, neovascularization of the cornea appears to only occurin association with an inflammatory cell infiltrate, and the degree ofangiogenesis is proportional to the extent of the inflammatory reaction.Corneal edema further facilitates blood vessel ingrowth by loosening thecorneal stromal framework and providing a pathway of “least resistance”through which the capillaries can grow.

Following the initial inflammatory reaction, capillary growth into thecornea proceeds in the same manner as it occurs in other tissues. Thenormally quiescent endothelial cells of the limbic capillaries andvenules are stimulated to divide and migrate. The endothelial cellsproject away from their vessels of origin, digest the surroundingbasement membrane and the tissue through which they will travel, andmigrate towards the source of the angiogenic stimulus. The blind endedsprouts acquire a lumen and then anastomose together to form capillaryloops. The end result is the establishment of a vascular plexus withinthe corneal stroma.

Anti-angiogenic factors and compositions of the present invention areuseful by blocking the stimulatory effects of angiogenesis promoters,reducing endothelial cell division, decreasing endothelial cellmigration, and impairing the activity of the proteolytic enzymessecreted by the endothelium.

Within particularly preferred embodiments of the invention, ananti-angiogenic factor may be prepared for topical administration insaline (combined with any of the preservatives and antimicrobial agentscommonly used in ocular preparations), and administered in eyedrop form.The anti-angiogenic factor solution or suspension may be prepared in itspure form and administered several times daily. Alternatively,anti-angiogenic compositions, prepared as described above, may also beadministered directly to the cornea. Within preferred embodiments, theanti-angiogenic composition is prepared with a muco-adhesive polymerwhich binds to cornea. Within further embodiments, the anti-angiogenicfactors or anti-angiogenic compositions may be utilized as an adjunct toconventional steroid therapy.

Topical therapy may also be useful prophylactically in comeal lesionswhich are known to have a high probability of inducing an angiogenicresponse (such as chemical bums). In these instances the treatment,likely in combination with steroids, may be instituted immediately tohelp prevent subsequent complications.

Within other embodiments, the anti-angiogenic compositions describedabove may be injected directly into the corneal stroma by anophthalmologist under microscopic guidance. The preferred site ofinjection may vary with the morphology of the individual lesion, but thegoal of the administration would be to place the composition at theadvancing front of the vasculature (i.e., interspersed between the bloodvessels and the normal cornea). In most cases this would involveperilimbic comeal injection to “protect” the cornea from the advancingblood vessels. This method may also be utilized shortly after a cornealinsult in order to prophylactically prevent corneal neovascularization.In this situation the material could be injected in the perilimbiccornea interspersed between the corneal lesion and its undesiredpotential limbic blood supply. Such methods may also be utilized in asimilar fashion to prevent capillary invasion of transplanted corneas.In a sustained-release form injections might only be required 2-3 timesper year. A steroid could also be added to the injection solution toreduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of an anti-angiogeniccomposition to the eye, such that the formation of blood vessels isinhibited.

Briefly, neovascular glaucoma is a pathological condition wherein newcapillaries develop in the iris of the eye. The angiogenesis usuallyoriginates from vessels located at the pupillary margin, and progressesacross the root of the iris and into the trabecular meshwork.Fibroblasts and other connective tissue elements are associated with thecapillary growth and a fibrovascular membrane develops which spreadsacross the anterior surface of the iris. Eventually this tissue reachesthe anterior chamber angle where it forms synechiae. These synechiae inturn coalesce, scar, and contract to ultimately close off the anteriorchamber angle. The scar formation prevents adequate drainage of aqueoushumor through the angle and into the trabecular meshwork, resulting inan increase in intraocular pressure that may result in blindness.

Neovascular glaucoma generally occurs as a complication of diseases inwhich retinal ischemia is predominant. In particular, about one third ofthe patients with this disorder have diabetic retinopathy and 28% havecentral retinal vein occlusion. Other causes include chronic retinaldetachment, end-stage glaucoma, carotid artery obstructive disease,retrolental fibroplasia, sickle-cell anemia, intraocular tumors, andcarotid cavernous fistulas. In its early stages, neovascular glaucomamay be diagnosed by high magnification slitlamp biomicroscopy, where itreveals small, dilated, disorganized capillaries (which leakfluorescein) on the surface of the iris. Later gonioscopy demonstratesprogressive obliteration of the anterior chamber angle by fibrovascularbands. While the anterior chamber angle is still open, conservativetherapies may be of assistance. However, once the angle closes surgicalintervention is required in order to alleviate the pressure.

Therefore, within one embodiment of the invention anti-angiogenicfactors (either alone or in an anti-angiogenic composition, as describedabove) maybe administered topically to the eye in order to treat earlyforms of neovascular glaucoma.

Within other embodiments of the invention, anti-angiogenic compositionsmay be implanted by injection of the composition into the region of theanterior chamber angle. This provides a sustained localized increase ofanti-angiogenic factor, and prevents blood vessel growth into the area.Implanted or injected anti-angiogenic compositions which are placedbetween the advancing capillaries of the iris and the anterior chamberangle can “defend” the open angle from neovascularization. Ascapillaries will not grow within a significant radius of theanti-angiogenic composition, patency of the angle could be maintained.Within other embodiments, the anti-angiogenic composition may also beplaced in any location such that the anti-angiogenic factor iscontinuously released into the aqueous humor. This would increase theanti-angiogenic factor concentration within the humor, which in turnbathes the surface of the iris and its abnormal capillaries, therebyproviding another mechanism by which to deliver the medication. Thesetherapeutic modalities may also be useful prophylactically and incombination with existing treatments.

Within another aspect of the present invention, methods are provided fortreating proliferative diabetic retinopathy, comprising the step ofadministering to a patient a therapeutically effective amount of ananti-angiogenic composition to the eyes, such that the formation ofblood vessels is inhibited.

Briefly, the pathology of diabetic retinopathy is thought to be similarto that described above for neovascular glaucoma. In particular,background diabetic retinopathy is believed to convert to proliferativediabetic retinopathy under the influence of retinal hypoxia. Generally,neovascular tissue sprouts from the optic nerve (usually within 10 mm ofthe edge), and from the surface of the retina in regions where tissueperfusion is poor. Initially the capillaries grow between the innerlimiting membrane of the retina and the posterior surface of thevitreous. Eventually, the vessels grow into the vitreous and through theinner limiting membrane. As the vitreous contracts, traction is appliedto the vessels, often resulting in shearing of the vessels and blindingof the vitreous due to hemorrhage. Fibrous traction from scarring in theretina may also produce retinal detachment.

The conventional therapy of choice is panretinal photocoagulation todecrease retinal tissue, and thereby decrease retinal oxygen demands.Although initially effective, there is a high relapse rate with newlesions forming in other parts of the retina. Complications of thistherapy include a decrease in peripheral vision of up to 50% ofpatients, mechanical abrasions of the cornea, laser-induced cataractformation, acute glaucoma, and stimulation of subretinal neovasculargrowth (which can result in loss of vision). As a result, this procedureis performed only when several risk factors are present, and therisk-benefit ratio is clearly in favor of intervention.

Therefore, within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection of ananti-angiogenic factor(s) (or anti-angiogenic composition) into theaqueous humor or the vitreous, in order to increase the localconcentration of anti-angiogenic factor in the retina. Preferably, thistreatment should be initiated prior to the acquisition of severe diseaserequiring photocoagulation. Within other embodiments of the invention,arteries which feed the neovascular lesions may be embolized (utilizinganti-angiogenic compositions, as described above)

Within another aspect of the present invention, methods are provided fortreating retrolental fibroblasia, comprising the step of administeringto a patient a therapeutically effective amount of an anti-angiogenicfactor (or anti-angiogenic composition) to the eye, such that theformation of blood vessels is inhibited.

Briefly, retrolental fibroblasia is a condition occurring in prematureinfants who receive oxygen therapy. The peripheral retinal vasculature,particularly on the temporal side, does not become fully formed untilthe end of fetal life. Excessive oxygen (even levels which would bephysiologic at term) and the formation of oxygen free radicals arethought to be important by causing damage to the blood vessels of theimmature retina. These vessels constrict, and then become structurallyobliterated on exposure to oxygen. As a result, the peripheral retinafails to vascularize and retinal ischemia ensues. In response to theischemia, neovascularization is induced at the junction of the normaland the ischemic retina.

In 75% of the cases these vessels regress spontaneously. However, in theremaining 25% there is continued capillary growth, contraction of thefibrovascular component, and traction on both the vessels and theretina. This results in vitreous hemorrhage and/or retinal detachmentwhich can lead to blindness. Neovascular angle-closure glaucoma is alsoa complication of this condition.

As it is often impossible to determine which cases will spontaneouslyresolve and which will progress in severity, conventional treatment(i.e., surgery) is generally initiated only in patients with establisheddisease and a well developed pathology. This “wait and see” approachprecludes early intervention, and allows the progression of disease inthe 25% who follow a complicated course. Therefore, within oneembodiment of the invention, topical administration of anti-angiogenicfactors (or anti-angiogenic compositions, as described above) may beaccomplished in infants which are at high risk for developing thiscondition in an attempt to cut down on the incidence of progression ofretrolental fibroplasia. Within other embodiments, intravitreousinjections and/or intraocular implants of an anti-angiogenic compositionmay be utilized. Such methods are particularly preferred in cases ofestablished disease, in order to reduce the need for surgery.

Other Therapeutic Uses of Anti-Angiogenic Compositions

Anti-angiogenic factors and compositions of the present invention may beutilized in a variety of additional methods in order to therapeuticallytreat a cancer or tumor. For example, anti-angiogenic factors orcompositions described herein may be formulated for topical delivery, inorder to treat cancers such as skin cancer, head and neck tumors, breasttumors, and Kaposi's sarcoma. Within yet other aspects, theanti-angiogenic factors or compositions provided herein may be utilizedto treat superficial forms of bladder cancer by, for example,intravesical administration.

In addition to cancer, however, numerous other non-tumorigenicangiogenesis-dependent diseases which are characterized by the abnormalgrowth of blood vessels may also be treated with the anti-angiogenicfactors or compositions of the present invention. Representativeexamples of such non-tumorigenic angiogenesis-dependent diseases includehypertrophic scars and keloids, proliferative diabetic retinopathy(discussed above), rheumatoid arthritis (discussed above), arteriovenousmalformations (discussed above), atherosclerotic plaques, delayed woundhealing, hemophilic joints, nonunion fractures, Osler-Weber syndrome,psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia(discussed above) and vascular adhesions.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering one of the above-described anti-angiogeniccompositions to a hypertrophic scar or keloid.

Briefly, healing of wounds and scar formation occurs in three phases:inflammation, proliferation, and maturation. The first phase,inflammation, occurs in response to an injury which is severe enough tobreak the skin. During this phase, which lasts 3 to 4 days, blood andtissue fluid form an adhesive coagulum and fibrinous network whichserves to bind the wound surfaces together. This is then followed by aproliferative phase in which there is ingrowth of capillaries andconnective tissue from the wound edges, and closure of the skin defect.Finally, once capillary and fibroblastic proliferation has ceased, thematuration process begins wherein the scar contracts and becomes lesscellular, less vascular, and appears flat and white. This final phasemay take between 6 and 12 months.

If too much connective tissue is produced and the wound remainspersistently cellular, the scar may become red and raised. If the scarremains within the boundaries of the original wound it is referred to asa hypertrophic scar, but if it extends beyond the original scar and intothe surrounding tissue, the lesion is referred to as a keloid.Hypertrophic scars and keloids are produced during the second and thirdphases of scar formation. Several wounds are particularly prone toexcessive endothelial and fibroblastic proliferation, including bums,open wounds, and infected wounds. With hypertrophic scars, some degreeof maturation occurs and gradual improvement occurs. In the case ofkeloids however, an actual tumor is produced which can become quitelarge. Spontaneous improvement in such cases rarely occurs.

Therefore, within one embodiment of the present invention eitheranti-angiogenic factors alone, or anti-angiogenic compositions asdescribed above, are directly injected into a hypertrophic scar orkeloid, in order to prevent the progression of these lesions. Thefrequency of injections will depend upon the release kinetics of thepolymer used (if present), and the clinical response. This therapy is ofparticular value in the prophylactic treatment of conditions which areknown to result in the development of hypertrophic scars and keloids(e.g., burns), and is preferably initiated after the proliferative phasehas had time to progress (approximately 14 days after the initialinjury), but before hypertrophic scar or keloid development.

As noted above, within yet another aspect of the present invention,vascular grafts are provided comprising a synthetic tube, the surface ofwhich is coated with an anti-angiogenic composition as described above.Briefly, vascular grafts are synthetic tubes, usually made of Dacron orGortex, inserted surgically to bypass arterial blockages, mostfrequently from the aorta to the femoral, or the femoral to thepopliteal artery. A major problem which particularly complicatesfemoral-popliteal bypass grafts is the formation of a subendothelialscar-like reaction in the blood vessel wall called neointimalhyperplasia, which narrows the lumen within and adjacent to either endof the graft, and which can be progressive. A graft coated with orcontaining anti-angiogenic factors (or anti-angiogenic compositions, asdescribed above) may be utilized to limit the formation of neointimalhyperplasia at either end of the graft. The graft may then be surgicallyplaced by conventional bypass techniques.

Anti-angiogenic compositions of the present invention may also beutilized in a variety of other manners. For example, they may beincorporated into surgical sutures in order to prevent stitchgranulomas, implanted in the uterus (in the same manner as an IUD) forthe treatment of menorrhagia or as a form of female birth control,administered as either a peritoneal lavage fluid or for peritonealimplantation in the treatment of endometriosis, attached to a monoclonalantibody directed against activated endothelial cells as a form ofsystemic chemotherapy, or utilized in diagnostic imaging when attachedto a radioactively labeled monoclonal antibody which recognizesactivated endothelial cells.

Formulation and Administration

As noted above, anti-angiogenic compositions of the present inventionmay be formulated in a variety of forms (e.g., microspheres, pastes,films or sprays). Further, the compositions of the present invention maybe formulated to contain more than one anti-angiogenic factor, tocontain a variety of additional compounds, to have certain physicalproperties (e.g., elasticity, a particular melting point, or a specifiedrelease rate). Within certain embodiments of the invention, compositionsmay be combined in order to achieve a desired effect (e.g., severalpreparations of microspheres may be combined in order to achieve both aquick and a slow or prolonged release of one or more anti-angiogenicfactor).

Anti-angiogenic factors and compositions of the present invention may beadministered either alone, or in combination with pharmaceutically orphysiologically acceptable carrier, excipients or diluents. Generally,such carriers should be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the therapeutic agent with buffers,antioxidants such as ascorbic acid, low molecular weight (less. thanabout 10 residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. Neutral bufferedsaline or saline mixed with nonspecific serum albumin are exemplaryappropriate diluents.

As noted above, anti-angiogenic factors, anti-angiogenic compositions,or pharmaceutical compositions provided herein may be prepared foradministration by a variety of different routes, including for exampleintrarticularly, intraocularly, intranasally, intradermally,sublingually, orally, topically, intravesically, intrathecally,topically, intravenously, intraperitoneally, intracranially,intramuscularly, subcutaneously, or even directly into a tumor ordisease site. Other representative routes of administration includegastroscopy, ECRP and colonoscopy, which do not require full operatingprocedures and hospitalization, but may require the presence of medicalpersonnel.

The anti-angiogenic factors, anti-angiogenic compositions andpharmaceutical compositions provided herein may be placed withincontainers, along with packaging material which provides instructionsregarding the use of such materials. Generally, such instructions willinclude a tangible expression describing the reagent concentration, aswell as within certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) which may benecessary to reconstitute the anti-angiogenic factor, anti-angiogeniccomposition, or pharmaceutical composition.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Preparation of Anti-Invasive Factor

The shoulder girdle and skull from a dogfish is excised, then scrapedwith a scalpel in order to remove all muscle and associated connectivetissue from the cartilage. The cartilage is then homogenized with atissue grinder, and extracted by continuous stirring at room temperaturefor 2 to 5 days in a solution containing 2.0 M guanidium hydrochlorideand 0.02 M MES at pH 6.0.

After 2 to 5 days, the cartilage extract is passed through gauze nettingin order to remove the larger constituents. The filtrate is then passedthrough an Amicon ultrafiltration unit which utilizes spiral-woundcartridges, with a molecular weight cutoff of 100,000. The filtrate(containing proteins with a molecular weight of less than 100,000daltons) is then dialyzed against 0.02 M MES buffer (pH 6) with anAmicon ultrafiltration unit which retains proteins with a molecularweight of greater than 3,000 daltons. Utilizing this method, lowmolecular weight proteins and constituents are removed, as well asexcessive amounts of guanidium HCl. The dialysate is concentrated to afinal concentration 9 mg/ml.

Example 2 Analysis of Various Agents for Anti-Angiogenic Activity

A. Chick Chorioallantoic Membrane (“Cam”) Assays Fertilized, domesticchick embryos were incubated for 3 days prior to shell-less culturing.In this procedure, the egg contents were emptied by removing the shelllocated around the air space. The interior shell membrane was thensevered and the opposite end of the shell was perforated to allow thecontents of the egg to gently slide out from the blunted end. The eggcontents were emptied into round-bottom sterilized glass bowls andcovered with petri dish covers. These were then placed into an incubatorat 90% relative humidity and 3% CO₂ and incubated for 3 days.

Paclitaxel (Sigma, St. Louis, Mo.) was mixed at concentrations of 1, 5,10, 30 μg per 10 ml aliquot of 0.5% aqueous methylcellulose. Sincepaclitaxel is insoluble in water, glass beads were used to produce fineparticles. Ten microliter aliquots of this solution were dried onparafilm for 1 hour forming disks 2 mm in diameter. The dried diskscontaining paclitaxel were then carefully placed at the growing edge ofeach CAM at day 6 of incubation. Controls were obtained by placingpaclitaxel-free methylcellulose disks on the CAMs over the same timecourse. After a 2 day exposure (day 8 of incubation) the vasculature wasexamined with the aid of a stereomicroscope. Liposyn II, a white opaquesolution, was injected into the CAM to increase the visibility of thevascular details. The vasculature of unstained, living embryos wereimaged using a Zeiss stereomicroscope which was interfaced with a videocamera (Dage-MTI Inc., Michigan City, Ind.). These video signals werethen displayed at 160 times magnification and captured using an imageanalysis system (Vidas, Kontron; Etching, Germany). Image negatives werethen made on a graphics recorder (Model 3000; Matrix Instruments,Orangeburg, N.Y.).

The membranes of the 8 day-old shell-less embryo were flooded with 2%glutaraldehyde in 0.1M Na cacodylate buffer; additional fixative wasinjected under the CAM. After 10 minutes in situ, the CAM was removedand placed into fresh fixative for 2 hours at room temperature. Thetissue was then washed overnight in cacodylate buffer containing 6%sucrose. The areas of interest were postfixed in 1% osmium tetroxide for1.5 hours at 4° C. The tissues were then dehydrated in a graded seriesof ethanols, solvent exchanged with propylene oxide, and embedded inSpurr resin. Thin sections were cut with a diamond knife, placed oncopper grids, stained, and examined in a Joel 1200EX electronmicroscope. Similarly, 0.5 mm sections were cut and stained with tolueneblue for light microscopy.

At day 11 of development, chick embryos were used for the corrosioncasting technique. Mercox resin (Ted Pella, Inc., Redding, Calif.) wasinjected into the CAM vasculature using a 30-gauge hypodermic needle.The casting material consisted of 2.5 grams of Mercox CL-2B polymer and0.05 grams of catalyst (55% benzoyl peroxide) having a 5 minutepolymerization time. After injection, the plastic was allowed to sit insitu for an hour at room temperature and then overnight in an oven at65° C. The CAM was then placed in 50% aqueous solution of sodiumhydroxide to digest all organic components. The plastic casts werewashed extensively in distilled water, air-dried, coated withgold/palladium, and viewed with the Philips 501B scanning electronmicroscope.

Results of the above experiments are shown in FIGS. 1-4. Briefly, thegeneral features of the normal chick shell-less egg culture are shown inFIG. 1A. At day 6 of incubation, the embryo is centrally positioned to aradially expanding network of blood vessels; the CAM develops adjacentto the embryo. These growing vessels lie close to the surface and arereadily visible making this system an idealized model for the study ofangiogenesis. Living, unstained capillary networks of the CAM can beimaged noninvasively with a stereomicroscope. FIG. 1B illustrates such avascular area in which the cellular blood elements within capillarieswere recorded with the use of a video/computer interface. The3-dimensional architecture of such CAM capillary networks is shown bythe corrosion casting method and viewed in the scanning electronmicroscope (FIG. 1C). These castings revealed underlying vessels whichproject toward the CAM surface where they form a single layer ofanastomotic capillaries.

Transverse sections through the CAM show an outer ectoderm consisting ofa double cell layer, a broader mesodermal layer containing capillarieswhich lie subjacent to the ectoderm, adventitial cells, and an inner,single endodernal cell layer (FIG. 1D). At the electron microscopiclevel, the typical structural details of the CAM capillaries aredemonstrated. Typically, these vessels lie in close association with theinner cell layer of ectoderm (FIG. 1E)

After 48 hours exposure to paclitaxel at concentrations of 0.25, 0.5, 1,5, 10, or 30 ug, each CAM was examined under living conditions with astereomicroscope equipped with a video/computer interface in order toevaluate the effects on angiogenesis. This imaging setup was used at amagnification of 160 times which permitted the direct visualization ofblood cells within the capillaries; thereby blood flow in areas ofinterest could be easily assessed and recorded. For this study, theinhibition of angiogenesis was defined as an area of the CAM lacking acapillary network and vascular blood flow. Throughout the experiments,avascular zones were assessed on a 4 point avascular gradient (Table I).This scale represents the degree of overall inhibition with maximalinhibition represented as a 3 on the avascular gradient scale.Paclitaxel was very consistent and induced a maximal avascular zone (6mm in diameter or a 3 on the avasculare gradient scale) within 48 hoursdepending on its concentration. TABLE I AVASCULAR GRADIENT 0 normalvascularity 1 lacking some microvascular movement 2* small avascularzone approximately 2 mm in diameter 3* avascularity extending beyond thedisk (6 mm in diameter)

The dose-dependent, experimental data of the effects of paclitaxel atdifferent concentrations are shown in Table II. TABLE II AngiogenicInhibition by Paclitaxel Paclitaxel in Methylcellulose Embryos EvaluatedDisks (μg) positive/total) % Inhibition 0.25 2/11 18 0.5 6/11 54 1 6/1540 5 20/27  76 10 16/21  76 30 31/31  100 0 0/40 0 (control)

TABLE III Angiogenic Inhibition of Paclitaxel-Loaded ThermopastePaclitaxel-loaded Embryos Evaluated Thermopaste (%) (positive/n)   0.254/4   0.5 4/4 1 8/8 5 4/4 10  5/5 20  6/6 0  0/30 (control)

Typical paclitaxel-treated CAMs are also shown with the transparentmethylcellulose disk centrally positioned over the avascular zonemeasuring 6 mm in diameter. At a slightly higher magnification, theperiphery of such avascular zones is clearly evident (FIG. 2C); thesurrounding functional vessels were often redirected away from thesource of paclitaxel (FIGS. 2C and 2D). Such angular redirecting ofblood flow was never observed under normal conditions. Another featureof the effects of paclitaxel was the formation of blood islands withinthe avascular zone representing the aggregation of blood cells.

The associated morphological alterations of the paclitaxel-treated CAMare readily apparent at both the light and electron microscopic levels.For the convenience of presentation, three distinct phases of generaltransition from the normal to the avascular state are shown. Near theperiphery of the avascular zone the CAM is hallmarked by an abundance ofmitotic cells within all three germ layers (FIGS. 3A and 4A). Thisenhanced mitotic division was also a consistent observation forcapillary endothelial cells. However, the endothelial cells remainedjunctionally intact with no extravasation of blood cells. With furtherdegradation, the CAM is characterized by the breakdown and dissolutionof capillaries (FIGS. 3B and 4B). The presumptive endothelial cells,typically arrested in mitosis, still maintain a close spatialrelationship with blood cells and lie subjacent to the ectoderm;however, these cells are not junctionally linked. The most centralportion of the avascular zone was characterized by a thickenedectodermal and endodermal layer (FIGS. 3C and 4C). Although these layerswere thickened, the cellular junctions remained intact and the layersmaintained their structural characteristics. Within the mesoderm,scattered mitotically arrested cells were abundant; these cells did notexhibit the endothelial cell polarization observed in the former phase.Also, throughout this avascular region, degenerating cells were commonas noted by the electron dense vacuoles and cellular debris (FIG. 4C).

In summary, this study demonstrated that 48 hours after paclitaxelapplication to the CAM, angiogenesis was inhibited. The blood vesselinhibition formed an avascular zone which was represented by threetransitional phases of paclitaxel's effect. The central, most affectedarea of the avascular zone contained disrupted capillaries withextravasated red blood cells; this indicated that intercellularjunctions between endothelial cells were absent. The cells of theendoderm and ectoderm maintained their intercellular junctions andtherefore these germ layers remained intact; however, they were slightlythickened. As the normal vascular area was approached, the blood vesselsretained their junctional complexes and therefore also remained intact.At the periphery of the paclitaxel-treated zone, further blood vesselgrowth was inhibited which was evident by the typical redirecting or“elbowing” effect of the blood vessels (FIG. 2D).

Paclitaxel-treated avascular zones also revealed an abundance of cellsarrested in mitosis in all three germ layers of the CAM; this was uniqueto paclitaxel since no previous study has illustrated such an event. Bybeing arrested in mitosis, endothelial cells could not undergo theirnormal metabolic functions involved in angiogenesis. In comparison, theavascular zone formed by suramin and cortisone acetate do not producemitotically arrested cells in the CAM; they only prevented further bloodvessel growth into the treated area. Therefore, even though these agentsare anti-angiogenic, there are many points in which the angiogenesisprocess may be targeted.

The effects of paclitaxel over the 48 hour duration were also observed.During this period of observation it was noticed that inhibition ofangiogenesis occurs as early as 9 hours after application. Histologicalsections revealed a similar morphology as seen in the first transitionphase of the avascular zone at 48 hours illustrated in FIGS. 3A and 4A.Also, we observed in the revascularization process into the avascularzone previously observed. It has been found that the avascular zoneformed by heparin and angiostatic steroids became revascularized 60hours after application. In one study, paclitaxel-treated avascularzones did not revascularize for at least 7 days after applicationimplying a more potent long-term effect.

Example 3 Encapsulation of Suramim

One milliliter of 5% ELVAX (poly(ethylene-vinyl acetate) cross-linkedwith 5% vinyl acetate) in dichloromethane (“DCM ”) is mixed with a fixedweight of sub-micron ground sodium suramin. This mixture is injectedinto 5 ml of 5% Polyvinyl Alcohol (“PVA”) in water in a 30 ml flatbottomed test tube. Tubes containing different weights of the drug arethen suspended in a multi-sample water bath at 40° for 90 minutes withautomated stirring. The mixtures are removed, and microsphere samplestaken for size analysis. Tubes are centrifuged at 1000 g for 5 min. ThePVA supematant is removed and saved for analysis (nonencapsulated drug).The microspheres are then washed (vortexed) in 5 ml of water andrecentrifuged. The 5 ml wash is saved for analysis (surface bound drug).Microspheres are then wetted in 50 ul of methanol, and vortexed in 1 mlof DCM to dissolve the ELVAX. The microspheres are then warmed to 40°C., and 5 ml of 50° C. water is slowly added with stirring. Thisprocedure results in the immediate evaporation of DCM, thereby causingthe release of sodium suramin into the 5 ml of water.

All samples were assayed for drug content by quantification offluorescence. Briefly, sodium suramin absorbs uv/vis with a lambda maxof 312 nm. This absorption is linear in the 0 to 100 ug/ml range in bothwater and 5% PVA. Sodium suramin also fluoresces strongly with anexcitation maximum at 312 nm, and emission maximum at 400 nm. Thisfluorescence is quantifiable in the 0 to 25 ug/ml range.

The results of these experiments is shown in FIGS. 5-11. Results areshown in FIGS. 5-10. Briefly, the size distribution of microspheres bynumber (FIG. 5) or by weight (FIG. 6) appears to be unaffected byinclusion of the drug in the DCM. Good yields of microspheres in the 20to 60 μm range may be obtained.

The encapsulation of suramin is very low (<1%) (see FIG. 8). However asthe weight of drug is increased in the DCM the total amount of drugencapsulated increased although the % encapsulation decreased. As isshown in FIG. 7, 50 ug of drug may be encapsulated in 50 mg of ELVAX.Encapsulation of sodium suramin in 2.5% PVA containing 10% NaCl is shownin FIG. 9 (size distribution by weight). Encapsulation of sodium suraminin 5% PVA containing 10% NaCl is shown in FIGS. 10 and 11 (sizedistribution by weight, and number, respectively).

To assess suramin and cortisone acetate as potential anti-angiogenicagents, each agent was mixed with 0.5% methylcellulose and applied thedried disks containing the agent onto the developing blood vessels ofthe 6-day old CAM. A combination treatment of suramin (70 μg) withcortisone acetate (20 μg) was successful in inhibiting angiogenesis whentested on the CAM for 48 hours. The resulting avascular region measured6 mm in diameter and revealed an absence of blood flow and theappearance of sparse blood islands (FIGS. 28A and 28B).

Example 4 Encapsulation of Paclitaxel

Five hundred micrograms of either paclitaxel or baccatin (a paclitaxelanalog, available from Inflazyme Pharmaceuticals Inc., Vancouver,British Columbia, Canada) are dissolved in 1 ml of a 50:50ELVAX:poly-l-lactic acid mixture in dcm. Microspheres are then preparedin a dissolution machine (Six-spindle dissolution tester, VanderKanp,Van Kell Industries Inc., U.S.A.) in triplicate at 200 rpm, 42° C., for3 hours. Microspheres so prepared are washed twice in water and sized onthe microscope.

Determination of paclitaxel encapsulation is undertaken in a uv/visassay (uv/vis lambda max. at 237 nm, fluorescence assay at excitation237, emission at 325 nm; Fluorescence results are presented in squarebrackets [ ]). Utilizing the procedures described above, 58 μg (±12 μg)[75 μg (±25 μg)] of paclitaxel may be encapsulated from a total 500 μgof starting material. This represents 12% (±2.4%) [15% (±5%)] of theoriginal weight, or 1.2% (±0.25%) [1.5% (±0.5%)] by weight of thepolymer. After 18 hours of tumbling in an oven at 37° C., 10.3% (±10%)[6% (±5.6%)] of the total paclitaxel had been released from themicrospheres.

For baccatin, 100±15 μg [83±231 g] of baccatin can be encapsulated froma total of 500 μg starting material. This represents a 20% (±3%) [17%(±5%) of the original weight of baccatin, and 2% (±0.3%) [1.7% (±0.5%)]by weight of the polymer. After 18 hours of tumbling in an oven at 37°C., 55% (±13%) [60% (±23%)] of the baccatin is released from themicrospheres.

Example 5 Analysis of Surgical Paste Containing Anti-AngiogenicCompositions

Fisher rats weighing approximately 300 grams are anesthetized, and a 1cm transverse upper abdominal incision is made. Two-tenths of amilliliter of saline containing 1×10⁶ live 9 L gliosarcoma cells (elutedimmediately prior to use from tissue culture) are injected into 2 of the5 hepatic lobes by piercing a 27 gauge needle 1 cm through the livercapsule. The abdominal wound is closed with 6.0 resorptible suture andskin clips and the GA terminated.

After 2 weeks, the tumor deposits will measure approximately 1 cm. Atthis time, both hepatic tumors are resected and the bare margin of theliver is packed with a hemostatic agent. The rats are divided into twogroups: half is administered polymeric carrier alone, and the other halfreceives an anti-angiogenic composition.

Rats are sacrificed 2, 7, 14, 21 and 84 days post hepatic resection. Inparticular, the rats are euthanized by injecting Euthanyl into thedorsal vein of the tail. The liver, spleen, and both lungs are removed,and histologic analysis is performed in order to study the tumors forevidence of anti-angiogenic activity.

Example 6 Embolization of Rat Arteries

Fisher rats weighing approximately 300 grams are anesthetized. Utilizingaseptic procedures, a 1 cm transverse upper abdominal incision is made,and the liver identified. Two-tenths of a milliliter of salinecontaining 1 million live 9 L gliosarcoma cells (eluted immediatelyprior from tissue culture) is injected into each of the 5 hepatic lobesby piercing a 27 gauge needle 1 cm through the liver capsule. One-tenthof a milliliter of normal saline is injected into the needle as it iswithdrawn to ensure that there is no spillage of cells into theperitoneal cavity. A pledget of gelfoam is placed on each of thepuncture sites to ensure hemostasis. The abdominal wound is closed with6.0 resorptible suture with skin clips, and the anesthetic terminated.The rat is returned to the animal care facility to have a standard dietfor 14 days, at which time each tumor deposit will measure 1 cm indiameter. The same procedure is repeated using Westar rats and a ColonCancer cell line (Radiologic Oncology Lab, M.D. Anderson, Houston,Tex.). In this instance, 3 weeks are required post-injection for thetumor deposits to measure 1 cm in diameter each.

After 2 or 3 weeks, depending on the rat species, the same generalanesthetic procedure is followed and a midline abdominal incision isperformed. The duodenum is flipped and the gastroduodenal artery isidentified and mobilized. Ties are placed above and below a cutdown siteon the midportion of the gastroduodenal artery (GDA), and 0.038 inchpolyethylene tubing is introduced in a retrograde fashion into theartery using an operating microscope. The tie below the insertion pointwill ligate the artery, while the one above will fix the catheter inplace. Angiography is performed by injecting 0.5 ml of 60% radiopaquecontrast material through the catheter as an x-ray is taken. The hepaticartery is then embolized by refluxing particles measuring 15-200 μmthrough the gastroduodenal artery catheter until flow, observed via theoperating microscope, is seen to cease for at least 30 seconds.Occlusion of the hepatic artery is confirmed by repeating an angiogramthrough the GDA catheter. Utilizing this procedure, one-half of the ratsreceive 15-200 μm particles of polymer alone, and the other half receive15-200 μm particles of the polymer-anti-angiogenic factor composition.The upper GDA ligature is tightened to occlude the GDA as the catheteris withdrawn to ensure hemostasis, and the hepatic artery (althoughembolized) is left intact. The abdomen is closed with 6.0 absorbablesuture and surgical clips.

The rats are subsequently sacrificed at 2, 7, 14, 21 and 84 dayspost-embolization in order to determine efficacy of the anti-angiogenicfactor. Briefly, general anesthetic is given, and utilizing asepticprecautions, a midline incision performed. The GDA is mobilized again,and after placing a ligature near the junction of the GDA and thehepatic artery (i.e., well above the site of the previous cutdown), a0.038-inch polyethylene tubing is inserted via cutdown of the vessel andangiography is performed. The rat is then euthanized by injectingEuthanyl into the dorsal vein of the tail. Once euthanasia is confirmed,the liver is removed en bloc along with the stomach, spleen and bothlungs.

Histologic analysis is performed on a prepared slide stained withhematoxylin and eosin (“H and E”) stain. Briefly, the lungs aresectioned at 1 cm intervals to assess passage of embolic materialthrough the hepatic veins and into the right side of circulation. Thestomach and spleen are also sectioned in order to assess inadvertentimmobilization from reflux of particles into the celiac access of thecollateral circulation.

Example 7 Transplantation of Biliary Stents in Rats

General anesthetic is administered to 300 gram Fisher rats. A 1 cmtransverse incision is then made in the upper abdomen, and the liveridentified. In the most superficial lobe, 0.2 ml of saline containing 1million cells of 9 L gliosarcoma cells (eluted from tissue cultureimmediately prior to use) is injected via a 27 gauge needle to a depthof 1 cm into the liver capsule. Hemostasis is achieved after removal ofthe needle by placing a pledget of gelfoam at the puncture sites. Salineis injected as the needle is removed to ensure no spillage of cells intothe peritoneal cavity or along the needle track. The general anestheticis terminated, and the animal returned to the animal care center andplaced on a normal diet.

Two weeks later, general anesthetic is administered, and utilizingaseptic precautions, the hepatic lobe containing the tumor is identifiedthrough a midline incision. A 16 gauge angiographic needle is theninserted through the hepatic capsule into the tumor, a 0.038-inchguidewire passed through the needle, and the needle withdrawn over theguidewire. A number 5 French dilator is passed over the guide into thetumor and withdrawn. A number 5 French delivery catheter is then passedover the wire containing a self-expanding stainless steel Wallstent (5mm in diameter and 1 cm long). The stent is deployed into the tumor andthe guidewire delivery catheter is removed. One-third of the rats have aconventional stainless steel stent inserted into the tumor, one-third astainless steel stent coated with polymer, and one third a stent coatedwith the polymer-anti-angiogenic factor compound. The general anestheticis terminated and the rat returned to the animal care facility.

A plain abdominal X-ray is performed at 2 days in order to assess thedegree of stent opening. Rats are sacrificed at 2, 7, 14, 28 and 56 dayspost-stent insertion by injecting Euthanyl, and their livers removed enbloc once euthanasia is confirmed. After fixation in formaldehyde for 48hours, the liver is sectioned at 0.5 mm intervals; including severingthe stent transversely using a fresh blade for each slice. Histologicsections stained with H and E are then analyzed to assess the degree oftumor ingrowth into the stent lumen.

Example 8 Manufacture of Microspheres

Equipment which is preferred for the manufacture of microspheresdescribed below include: 200 ml water jacketed beaker (Kimax or Pyrex),Haake circulating water bath, overhead stirrer and controller with 2inch diameter (4 blade, propeller type stainless steel stirrer—Fisherbrand), 500 ml glass beaker, hot plate/stirrer (Corning brand), 4×50 mlpolypropylene centrifuge tubes (Nalgene), glass scintillation vials withplastic insert caps, table top centrifuge (GPR Beckman), high speedcentrifuge—floor model (JS 21 Beckman), Mettler analytical balance (AJ100, 0.1 mg), Mettler digital top loading balance (AE 163, 0.01 mg),automatic pipetter (Gilson). Reagents include Polycaprolactone(“PCL”—mol wt 10,000 to 20,000; Polysciences, Warrington Pa., USA),“washed” (see later method of “washing”) Ethylene Vinyl Acetate (“EVA”),Poly(DL)lactic acid (“PLA”—mol wt 15,000 to 25,000; Polysciences),Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000; 99% hydrolyzed;Aldrich Chemical Co., Milwaukee Wis., USA), Dichloromethane (“DCM” or“methylene chloride”; HPLC grade Fisher scientific), and distilledwater.

A. Preparation of 5% (w/v) Polymer Solutions

Depending on the polymer solution being prepared, 1.00 g of PCL or PLA,or 0.50 g each of PLA and washed EVA is weighed directly into a 20 mlglass scintillation vial. Twenty milliliters of DCM is then added, andthe vial tightly capped. The vial is stored at room temperature (25° C.)for one hour (occasional shaking may be used), or until all the polymerhas dissolved (the solution should be clear). The solution may be storedat room temperature for at least two weeks.

B. Preparation of 5% (w/v) Stock Solution of PVA

Twenty-five grams of PVA is weighed directly into a 600 ml glass beaker.Five hundred milliliters of distilled water is added, along with a 3inch Teflon coated stir bar. The beaker is covered with glass todecrease evaporation losses, and placed into a 2000 ml glass beakercontaining 300 ml of water (which acts as a water bath). The PVA isstirred at 300 rpm at 85° C. (Corning hot plate/stirrer) for 2 hours oruntil fully dissolved. Dissolution of the PVA may be determined by avisual check; the solution should be clear. The solution is thentransferred to a glass screw top storage container and stored at 4° C.for a maximum of two months. The solution, however should be warmed toroom temperature before use or dilution.

C. Procedure for Producing Microspheres

Based on the size of microspheres being made (see Table 1), 100 ml ofthe PVA solution (concentrations given in Table IV) is placed into the200 ml water jacketed beaker. Haake circulating water bath is connectedto this beaker and the contents are allowed to equilibrate at 27° C.(±10° C.) for 10 minutes. Based on the size of microspheres being made(see Table IV), the start speed of the overhead stirrer is set, and theblade of the overhead stirrer placed half way down in the PVA solution.The stirrer is then started, and 10 ml of polymer solution (polymersolution used based on type of microspheres being produced) is thendripped into the stirring PVA over a period of 2 minutes using a 5 mlautomatic pipetter. After 3 minutes the stir speed is adjusted (seeTable IV), and the solution stirred for an additional 2.5 hours. Thestirring blade is then removed from the microsphere preparation, andrinsed with 10 ml of distilled water so that the rinse solution drainsinto the microsphere preparation. The microsphere preparation is thenpoured into a 500 ml beaker, and the jacketed water bath washed with 70ml of distilled water, which is also allowed to drain into themicrosphere preparation. The 180 ml microsphere preparation is thenstirred with a glass rod, and equal amounts are poured into fourpolypropylene 50 ml centrifuge tubes. The tubes are then capped, andcentrifuged for 10 minutes (force given in Table III). A 5 ml automaticpipetter or vacuum suction is then utilized to draw 45 ml of the PVAsolution off of each microsphere pellet. TABLE III PVA concentrations,stir speeds, and centrifugal force requirements for each diameter rangeof microspheres. MICROSPHERE DIAMETER RANGES PRODUCTION 30 μm to 10 μmto 0.1 μm to STAGE 100 μm 30 μm 3 μm PVA 2.5% (w/v) 5% (w/v) (i.e., 3.5%(w/v) concentration (i.e.,) dilute undiluted (i.e., dilute 5% stockstock) 5% stock with distilled with distilled water water Starting 500rpm +/− 500 rpm +/− 3000 rpm +/− Stir Speed 50 rpm 50 rpm 200 rpmAdjusted 500 rpm +/− 500 rpm+/− 2500 rpm +/− Stir Speed 50 rpm 50 rpm200 rpm Centrifuge 1000 g +/− 1000 g +/− 10 000 g +/− Force 100 g 100 g1000 g (Table top (Table top (High speed model) model) model)

Five milliliters of distilled water is then added to each centrifugetube, which is then vortexed to resuspend the microspheres. The fourmicrosphere suspensions are then pooled into one centrifuge tube alongwith 20 ml of distilled water, and centrifuged for another 10 minutes(force given in Table 1). This process is repeated two additional timesfor a total of three washes. The microspheres are then centrifuged afinal time, and resuspended in 10 ml of distilled water. After the finalwash, the microsphere preparation is transferred into a preweighed glassscintillation vial. The vial is capped, and left overnight at roomtemperature (25° C.) in order to allow the microspheres to sediment outunder gravity. Microspheres which fall in the size range of 0.1 um to 3um do not sediment out under gravity, so they are left in the 10 mlsuspension.

D. Drying of 10 μm to 30 μm or 30 μm to 100 μm Diameter Microspheres

After the microspheres have sat at room temperature overnight, a 5 mlautomatic pipetter or vacuum suction is used to draw the supernatant offof the sedimented microspheres. The microspheres are allowed to dry inthe uncapped vial in a drawer for a period of one week or until they arefully dry (vial at constant weight). Faster drying may be accomplishedby leaving the uncapped vial under a slow stream of nitrogen gas (flowapprox. 10 ml/min.) in the fume hood. When fully dry (vial at constantweight), the vial is weighed and capped. The labeled, capped vial isstored at room temperature in a drawer. Microspheres are normally storedno longer than 3 months.

E. Drying of 0.1 μm to 3 μm Diameter Microspheres

This size range of microspheres will not sediment out, so they are leftin suspension at 4° C. for a maximum of four weeks. To determine theconcentration of microspheres in the 10 ml suspension, a 200 μl sampleof the suspension is pipetted into a 1.5 ml preweighed microfuge tube.The tube is then centrifuged at 10,000 g (Eppendorf table topmicrofuge), the supernatant removed, and the tube allowed to dry at 50°C. overnight. The tube is then reweighed in order to determine theweight of dried microspheres within the tube.

F. Manufacture of Paclitaxel Loaded Microsphere

In order to prepare paclitaxel containing microspheres, an appropriateamount of weighed paclitaxel (based upon the percentage of paclitaxel tobe encapsulated) is placed directly into a 20 ml glass scintillationvial. Ten milliliters of an appropriate polymer solution is then addedto the vial containing the paclitaxel, which is then vortexed until thepaclitaxel has dissolved.

Microspheres containing paclitaxel may then be produced essentially asdescribed above in steps (C) through (E).

Example 9 Manufacture of Stent Coating

Reagents and equipment which are utilized within the followingexperiments include (medical grade stents obtained commercially from avariety of manufacturers; e.g., the “Strecker” stent) and holdingapparatus, 20 ml glass scintillation vial with cap (plastic inserttype), TLC atomizer, Nitrogen gas tank, glass test tubes (various sizesfrom 1 ml and up), glass beakers (various sizes), Pasteur. pipette,tweezers, Polycaprolactone (“PCL”—mol wt 10,000 to 20,000;Polysciences), Paclitaxel (Sigma Chemical Co., St. Louis, Mo., 95%purity), Ethylene vinyl acetate (“EVA”—washed—see previous),Poly(DL)lactic acid (“PLA”—mol wt 15,000 to 25,000; Polysciences),dichloromethane (“DCM”—HPLC grade, Fisher Scientific).

A. Procedure for Sprayed Stents

The following describes a typical method using a 3 mm crimped diameterinterleaving metal wire stent of approximately 3 cm length. For largerdiameter stents, larger volumes of polymer/drug solution are used.

Briefly, a sufficient quantity of polymer is weighed directly into a 20ml glass scintillation vial, and sufficient DCM added in order toachieve a 2% w/v solution. The vial is then capped and mixed by hand inorder to dissolve the polymer. The stent is then assembled in a verticalorientation, tying the stent to a retort stand with nylon. Position thisstent holding apparatus 6 to 12 inches above the fume hood floor on asuitable support (e.g., inverted 2000 ml glass beaker) to enablehorizontal spraying. Using an automatic pipette, a suitable volume(minimum 5 ml) of the 2% polymer solution is transferred to a separate20 ml glass scintillation vial. An appropriate amount of paclitaxel isthen added to the solution and dissolved by hand shaking.

To prepare for spraying, remove the cap of this vial and dip the barrel(only) of an TLC atomizer into the polymer solution. Note that thereservoir of the atomizer need not be used in this procedure: the 20 mlglass vial acts as a reservoir. Connect the nitrogen tank to the gasinlet of the atomizer. Gradually increase the pressure until atomizationand spraying begins. Note the pressure and use this pressure throughoutthe procedure. To spray the stent use 5 second oscillating sprays with a15 second dry time between sprays. After 5 sprays, rotate the stent 90°and spray that portion of the stent. Repeat until all sides of the stenthave been sprayed. During the dry time, finger crimp the gas line toavoid wastage of the spray. Spraying is continued until a suitableamount of polymer is deposited on the stents. The amount may be based onthe specific stent application in vivo. To determine the amount, weighthe stent after spraying has been completed and the stent has dried.Subtract the original weight of the stent from the finished weight andthis produces the amount of polymer (plus paclitaxel) applied to thestent. Store the coated stent in a sealed container.

B. Procedure for Dipped Stents

The following describes a typical method using a 3 mm crimped diameterinterleaving metal wire stent of approximately 3 cm length. For largerdiameter stents, larger volumes of polymer/drug solution are used inlarger sized test tubes.

Weigh 2 g of EVA into a 20 ml glass scintillation vial and add 20 ml ofDCM. Cap the vial and leave it for 2 hours to dissolve (hand shake thevial frequently to assist the dissolving process). Weigh a known weightof paclitaxel directly into a 1 ml glass test tube and add 0.5 ml of thepolymer solution. Using a glass Pasteur pipette, dissolve the paclitaxelby gently pumping the polymer solution. Once the paclitaxel isdissolved, hold the test tube in a near horizontal position (the stickypolymer solution will not flow out). Using tweezers, insert the stentinto the tube all the way to the bottom. Allow the polymer solution toflow almost to the mouth of the test tube by angling the mouth belowhorizontal and then restoring the test tube to an angle slightly abovethe horizontal. While slowly rotating the stent in the tube, slowlyremove the stent (approximately 30 seconds).

Hold the stent in a vertical position to dry. Some of the sealedperforations may pop so that a hole exists in the continuous sheet ofpolymer. This may be remedied by repeating the previous dippingprocedure, however repetition of the procedure can also lead to furtherpopping and a general uneven build up of polymer. Generally, it isbetter to dip the stent just once and to cut out a section of stent thathas no popped perforations. Store the dipped stent in a sealedcontainer.

Example 10 Manufacture of Surgical “Pastes”

As noted above, the present invention provides a variety ofpolymeric-containing drug compositions that may be utilized within avariety of clinical situations. For example, compositions may beproduced: (1) as a “thermopaste” that is applied to a desired site as afluid, and hardens to a solid of the desired shape at a specifiedtemperature (e.g., body temperature); (2) as a spray (i.e., “nanospray”)which may delivered to a desired site either directly or through aspecialized apparatus (e.g., endoscopy), and which subsequently hardensto a solid which adheres to the tissue to which it is applied; (3) as anadherent, pliable, resilient, angiogeneis inhibitor-polymer film appliedto a desired site either directly or through a specialized apparatus,and which preferably adheres to the site to which it is applied; and (4)as a fluid composed of a suspension of microspheres in an appropriatecarrier medium, which is applied to a desired site either directly orvia a specialized apparatus, and which leaves a layer of microspheres atthe application site. Representative examples of each of the aboveembodiments is set forth in more detail below.

A. Procedure for Producing Thermopaste

Reagents and equipment which are utilized within the followingexperiments include a sterile glass syringe (1 ml), Corning hotplate/stirrer, 20 ml glass scintillation vial, moulds (e.g., 50 μl DSCpan or 50 ml centrifuge tube cap inner portion), scalpel and tweezers,Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), and Paclitaxel (Sigma grade 95% purity minimum).

Weigh 5.00 g of polycaprolactone directly into a 20 ml glassscintillation vial. Place the vial in a 600 ml beaker containing 50 mlof water. Gently heat the beaker to 65° C. and hold it at thattemperature for 20 minutes. This allows the polymer to melt. Thoroughlymix a known weight of paclitaxel, or other angiogenesis inhibitor intothe melted polymer at 65° C. Pour the melted polymer into a prewarmed(60° C. oven) mould. Use a spatula to assist with the pouring process.Allow the mould to cool so the polymer solidifies. Cut or break thepolymer into small pieces (approximately 2 mm by 2 mm in size). Thesepieces must fit into a 1 ml glass syringe. Remove the plunger from the 1ml glass syringe (do not remove the cap from the tip) and place it on abalance. Zero the balance.

Weigh 0.5 g of the pieces directly into the open end of the syringe.Place the glass syringe upright (capped tip downwards) into a 500 mlglass beaker containing distilled water at 65° C. (corning hot plate) sothat no water enters the barrel. The polymer melts completely within 10minutes in this apparatus. When the polymer pieces have melted, removethe barrel from the water bath, hold it horizontally and remove the cap.Insert the plunger into the barrel and compress the melted polymer intoa sticky mass at the tip end of the barrel. Cap the syringe and allow itto cool to room temperature.

For application, the syringe may be reheated to 60° C. and administeredas a liquid which solidifies when cooled to body temperature.

B. Procedure for Producing Nanospray

Nanospray is a suspension of small microspheres in saline. If themicrospheres are very small (i.e., under 1 μm in diameter) they form acolloid so that the suspension will not sediment under gravity. As isdescribed in more detail below, a suspension of 0.1 μm to 1 μmmicroparticles may be created suitable for deposition onto tissuethrough a finger pumped aerosol. Equipment and materials which may beutilized to produce nanospray include 200 ml water jacketed beaker(Kimax or Pyrex), Haake circulating water bath, overhead stirrer andcontroller with 2 inch diameter (4 blade, propeller type stainless steelstirrer; Fisher brand), 500 ml glass beaker, hot plate/stirrer (Corningbrand), 4×50 ml polypropylene centrifuge tubes (Nalgene), glassscintillation vials with plastic insert caps, table top centrifuge(Beckman), high speed centrifuge—floor model (JS 21 Beckman), Mettleranalytical balance (AJ 100, 0.1 mg), Mettler digital top loading balance(AE 163, 0.01 mg), automatic pipetter (Gilson), sterile pipette tips,pump action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood,Polycaprolactone (“PCL”—mol wt 10,000 to 20,000; Polysciences,Warrington, Pa. USA), “washed” (see previous) Ethylene Vinyl Acetate(“EVA”), Poly(DL)lactic acid (“PLA” mol wt 15,000 to 25,000;Polysciences), Polyvinyl Alcohol (“PVA”—mol wt 124,000 to 186,000; 99%hydrolyzed; Aldrich Chemical Co., Milwaukee, Wis. USA), Dichloromethane(“DCM” or “methylene chloride;” HPLC grade Fisher scientific), Distilledwater, sterile saline (Becton and Dickenson or equivalent)

1. Preparation of 5% (w/v) Polymer Solutions Depending on the polymersolution being prepared, weigh 1.00 g of PCL or PLA or 0.50 g each ofPLA and washed EVA directly into a 20 ml glass scintillation vial. Usinga measuring cylinder, add 20 ml of DCM and tightly cap the vial. Leavethe vial at room temperature (25° C.) for one hour or until all thepolymer has dissolved (occasional hand shaking may be used). Dissolvingof the polymer can be determined by a visual check; the solution shouldbe clear. Label the vial with the name of the solution and the date itwas produced. Store the solutions at room temperature and use within twoweeks.

2. Preparation of 3.5% (w/v) Stock Solution of PVA

The solution can be prepared by following the procedure given below, orby diluting the 5% (w/v) PVA stock solution prepared for production ofmicrospheres (see Example 8). Briefly, 17.5 g of PVA is weighed directlyinto a 600 ml glass beaker, and 500 ml of distilled water is added.Place a 3 inch Teflon coated stir bar in the beaker. Cover the beakerwith a cover glass to reduce evaporation losses. Place the beaker in a2000 ml glass beaker containing 300 ml of water. This will act as awater bath. Stir the PVA at 300 rpm at 85° C. (Corning hotplate/stirrer) for 2 hours or until fully dissolved. Dissolving of thePVA can be determined by a visual check; the solution should be clear.Use a pipette to transfer the solution to a glass screw top storagecontainer and store at 4° C. for a maximum of two months. This solutionshould be warmed to room temperature before use or dilution.

3. Procedure for Producing Nanospray

Place the stirring assembly in a fume hood. Place 100. ml of the 3.5%PVA solution in the 200 ml water jacketed beaker. Connect the Haakewater bath to this beaker and allow the contents to equilibrate at 27°C. (±1° C.) for 10 minutes. Set the start speed of the overhead stirrerat 3000 rpm (±200 rpm). Place the blade of the overhead stirrer half waydown in the PVA solution and start the stirrer. Drip 10 ml of polymersolution (polymer solution used based on type of nanospray beingproduced) into the stirring PVA over a period of 2 minutes using a 5 mlautomatic pipetter. After 3 minutes, adjust the stir speed to 2500 rpm(±200 rpm) and leave the assembly for 2.5 hours. After 2.5 hours, removethe stirring blade from the nanospray preparation and rinse with 10 mlof distilled water. Allow the rinse solution to go into the nanospraypreparation.

Pour the microsphere preparation into a 500 ml beaker. Wash the jacketedwater bath with 70 ml of distilled water. Allow the 70 ml rinse solutionto go into the microsphere preparation. Stir the 180 ml microspherepreparation with a glass rod and pour equal amounts of it into fourpolypropylene 50 ml centrifuge tubes. Cap the tubes. Centrifuge thecapped tubes at 10 000 g (±1000 g) for 10 minutes. Using a 5 mlautomatic pipetter or vacuum suction, draw 45 ml of the PVA solution offof each microsphere pellet and discard it. Add 5 ml of distilled waterto each centrifuge tube and use a vortex to resuspend the microspheresin each tube. Using 20 ml of distilled water, pool the four microspheresuspensions into one centrifuge tube. To wash the microspheres,centrifuge the nanospray preparation for 10 minutes at 10 000 g (±1000g). Draw the supernatant off of the microsphere pellet. Add 40 ml ofdistilled water and use a vortex to resuspend the microspheres. Repeatthis process two more times for a total of three washes. Do a fourthwash but use only 10 ml (not 40 ml) of distilled water when resuspendingthe microspheres. After the fourth wash, transfer the microspherepreparation into a preweighed glass scintillation vial.

Cap the vial and let it to sit for 1 hour at room temperature (25° C.)to allow the 2 μm and 3 μm diameter microspheres to sediment out undergravity. After 1 hour, draw off the top 9 ml of suspension using a 5 mlautomatic pipetter. Place the 9 ml into a sterile capped 50 mlcentrifuge tube. Centrifuge the suspension at 10 000 g (±1000 g) for 10minutes. Discard the supernatant and resuspend the pellet in 20 ml ofsterile saline. Centrifuge the suspension at 10 000 g (±1000 g) for 10minutes. Discard the supernatant and resuspend the pellet in sterilesaline. The quantity of saline used is dependent on the final requiredsuspension concentration (usually 10% w/v). Thoroughly rinse the aerosolapparatus in sterile saline and add the nanospray suspension to theaerosol.

C. Manufacture of Paclitaxel Loaded Nanospray

To manufacture nanospray containing paclitaxel, use Paclitaxel (Sigmagrade 95% purity). To prepare the polymer drug stock solution, weigh theappropriate amount of paclitaxel directly into a 20 ml glassscintillation vial. The appropriate amount is determined based on thepercentage of paclitaxel to be in the nanospray. For example, ifnanospray containing 5% paclitaxel was required, then the amount ofpaclitaxel weighed would be 25 mg since the amount of polymer added is10 ml of a 5% polymer in DCM solution (see next step).

Add 10 ml of the appropriate 5% polymer solution to the vial containingthe paclitaxel. Cap the vial and vortex or hand swirl it to dissolve thepaclitaxel (visual check to ensure paclitaxel dissolved). Label the vialwith the date it was produced. This is to be used the day it isproduced.

Follow the procedures as described above, except that polymer/drug(e.g., paclitaxel) stock solution is substituted for the polymersolution.

D. Procedure for Producing Film

The term film refers to a polymer formed into one of many geometricshapes. The film may be a thin, elastic sheet of polymer or a 2 mm thickdisc of polymer. This film is designed to be placed on exposed tissue sothat any encapsulated drug is released from the polymer over a longperiod of time at the tissue site. Films may be made by severalprocesses, including for example, by casting, and by spraying.

In the casting technique, polymer is either melted and poured into ashape or dissolved in dichloromethane and poured into a shape. Thepolymer then either solidifies as it cools or solidifies as the solventevaporates, respectively. In the spraying technique, the polymer isdissolved in solvent and sprayed onto glass, as the solvent evaporatesthe polymer solidifies on the glass. Repeated spraying enables a buildup of polymer into a film that can be peeled from the glass.

Reagents and equipment which were utilized within these experimentsinclude a small beaker, Corning hot plate stirrer, casting moulds (e.g.,50 ml centrifuge tube caps) and mould holding apparatus, 20 ml glassscintillation vial with cap (Plastic insert type), TLC atomizer,Nitrogen gas tank, Polycaprolactone (“PCL”—mol wt 10,000 to 20,000;Polysciences), Paclitaxel (Sigma 95% purity), Ethanol, “washed” (seeprevious) Ethylene vinyl acetate (“EVA”), Poly(DL)lactic acid (“PLA”—molwt 15,000 to 25,000; Polysciences), Dichloromethane (HPLC grade FisherScientific).

1. Procedure for Producing Films—Melt Casting

Weigh a known weight of PCL directly into a small glass beaker. Placethe beaker in a larger beaker containing water (to act as a water bath)and put it on the hot plate at 70° C. for 15 minutes or until thepolymer has fully melted. Add a known weight of drug to the meltedpolymer and stir the mixture thoroughly. To aid dispersion of the drugin the melted PCL, the drug may be suspended/dissolved in a small volume(<10% of the volume of the melted PCL) of 100% ethanol. This ethanolsuspension is then mixed into the melted polymer. Pour the meltedpolymer into a mould and let it to cool. After cooling, store the filmin a container.

2. Procedure for Producing Films—Solvent Casting

Weigh a known weight of PCL directly into a 20 ml glass scintillationvial and add sufficient DCM to achieve a 10% w/v solution. Cap the vialand mix the solution. Add sufficient paclitaxel to the solution toachieve the desired final paclitaxel concentration. Use hand shaking orvortexing to dissolve the paclitaxel in the solution. Let the solutionsit for one hour (to diminish the presence of air bubbles) and then pourit slowly into a mould. The mould used is based on the shape required.Place the mould in the fume hood overnight. This will allow the DCM toevaporate. Either leave the film in the mould to store it or peel it outand store it in a sealed container.

3. Procedure for Producing Films—Sprayed

Weigh sufficient polymer directly into a 20 ml glass scintillation vialand add sufficient DCM to achieve a 2% w/v solution. Cap the vial andmix the solution to dissolve the polymer (hand shaking). Assemble themoulds in a vertical orientation in a suitable mould holding apparatusin the fume hood. Position this mould holding apparatus 6 to 12 inchesabove the fume hood floor on a suitable support (e.g., inverted 2000 mlglass beaker) to enable horizontal spraying. Using an automatic pipette,transfer a suitable volume (minimum 5 ml) of the 2% polymer solution toa separate 20 ml glass scintillation vial. Add sufficient paclitaxel tothe solution and dissolve it by hand shaking the capped vial. To preparefor spraying, remove the cap of this vial and dip the barrel (only) ofan TLC atomizer into the polymer solution. Note: the reservoir of theatomizer is not used in this procedure—the 20 ml glass vial acts as areservoir.

Connect the nitrogen tank to the gas inlet of the atomizer. Graduallyincrease the pressure until atomization and spraying begins. Note thepressure and use this pressure throughout the procedure. To spray themoulds use 5 second oscillating sprays with a 15 second dry time betweensprays. During the dry time, finger crimp the gas line to avoid wastageof the spray. Spraying is continued until a suitable thickness ofpolymer is deposited on the mould. The thickness is based on therequest. Leave the sprayed films attached to the moulds and store insealed containers.

E. Procedure for Producing Nanopaste

Nanopaste is a suspension of microspheres suspended in a hydrophilicgel. Within one aspect of the invention, the gel or paste can be smearedover tissue as a method of locating drug loaded microspheres close tothe target tissue. Being water based, the paste will soon become dilutedwith bodily fluids causing a decrease in the stickiness of the paste anda tendency of the microspheres to be deposited on nearby tissue. A poolof microsphere encapsulated drug is thereby located close to the targettissue.

Reagents and equipment which were utilized within these experimentsinclude glass beakers, Carbopol 925 (pharmaceutical grade, GoodyearChemical Co.), distilled water, sodium hydroxide (1 M) in watersolution, sodium hydroxide solution (5 M) in water solution,microspheres in the 0.1 lm to 3 lm size range suspended in water at 20%w/v (See previous).

1. Preparation of 5% w/v Carbopol Gel

Add a sufficient amount of carbopol to 1 M sodium hydroxide to achieve a5% w/v solution. To dissolve the carbopol in the 1 M sodium hydroxide,allow the mixture to sit for approximately one hour. During this timeperiod, stir the mixture using a glass rod. After one hour, take the pHof the mixture. A low pH indicates that the carbopol is not fullydissolved. The pH you want to achieve is 7.4.

Use 5 M sodium hydroxide to adjust the pH. This is accomplished byslowly adding drops of 5 M sodium hydroxide to the mixture, stirring themixture and taking the pH of the mixture. It usually takes approximatelyone hour to adjust the pH to 7.4. Once a pH of 7.4 is achieved, coverthe gel and let it sit for 2 to 3 hours. After this time period, checkthe pH to ensure it is still at 7.4. If it has changed, adjust back topH 7.4 using 5 M sodium hydroxide. Allow the gel to sit for a few hoursto ensure the pH is stable at 7.4. Repeat the process until the desiredpH is achieved and is stable. Label the container with the name of thegel and the date. The gel is to be used to make nanopaste within thenext week.

2. Procedure for Producing Nanopaste

Add sufficient 0.1 μm to 3 μm microspheres to water to produce a 20%suspension of the microspheres. Put 8 ml of the 5% w/v carbopol gel in aglass beaker. Add 2 ml of the 20% microsphere suspension to the beaker.Using a glass rod or a mixing spatula, stir the mixture to thoroughlydisperse the microspheres throughout the gel. This usually takes 30minutes. Once the microspheres are dispersed in the gel, place themixture in a storage jar. Store the jar at 4° C. It must be used withina one month period.

Example 11 Controlled Delivery of Paclitaxel from Microspheres Composedof a Blend of Ethylene-Vinyl-Acetate Copolymer and Poly (D,L LacticAcid). In vivo Testing of the Microspheres on the CAM Assay

This example describes the preparation of paclitaxel-loaded microspherescomposed of a blend of biodegradable poly (d,l-lactic acid) (PLA)polymer and nondegradable ethylene-vinyl acetate (EVA) copolymer. Inaddition, the in vitro release rate and anti-angiogenic activity ofpaclitaxel released from microspheres placed on a CAM are demonstrated.

Reagents which were utilized in these experiments include paclitaxel,which is purchased from Sigma Chemical Co. (St. Louis, Mo.); PLA(molecular weight 15,000-25,000) and EVA (60% vinyl acetate) (purchasedfrom Polysciences (Warrington, Pa.); polyvinyl alcohol (PVA) (molecularweight 124,000-186,000, 99% hydrolysed, purchased from Aldrich ChemicalCo. (Milwaukee, Wis.)) and Dichloromethane (DCM) (HPLC grade, obtainedfrom Fisher Scientific Co). Distilled water is used throughout.

A. Preparation of Microspheres

Microspheres are prepared essentially as described in Example 8utilizing the solvent evaporation method. Briefly, 5% w/v polymersolutions in 20 mL DCM are prepared using blends of EVA:PLA between35:65 to 90:10. To 5 mL of 2.5% w/v PVA in water in a 20 mL glass vialis added 1 mL of the polymer solution dropwise with stirring. Sixsimilar vials are assembled in a six position overhead stirrer,dissolution testing apparatus (Vanderkamp) and stirred at 200 rpm. Thetemperature of the vials is increased from room temperature to 40° C.over 15 min and held at 40° C. for 2 hours. Vials are centrifuged at.500×g and the microspheres washed three times in water. At some EVA:PLApolymer blends, the microsphere samples aggregated during the washingstage due to the removal of the dispersing or emulsifying agent, PVA.This aggregation effect could be analyzed semi-quantitatively sinceaggregated microspheres fused and the fused polymer mass floated on thesurface of the wash water. This surface polymer layer is discardedduring the wash treatments and the remaining, pelleted microspheres areweighed. The % aggregation is determined from${\%\quad{aggregation}} = \frac{1 - {\left( {{weight}\quad{of}\quad{pelleted}\quad{microspheres}} \right) \times 100}}{{initial}\quad{polymer}\quad{weight}}$

Paclitaxel loaded microspheres (0.6% w/w paclitaxel) are prepared bydissolving the paclitaxel in the 5% w/v polymer solution in DCM. Thepolymer blend used is 50:50 EVA:PLA. A “large” size fraction and “small”size fraction of microspheres are produced by adding thepaclitaxel/polymer solution dropwise into 2.5% w/v PVA and 5% w/v PVA,respectively. The dispersions are stirred at 40° C. at 200 rpm for 2hours, centrifuged and washed 3 times in water as described previously.Microspheres are air dried and samples are sized using an opticalmicroscope with a stage micrometer. Over 300 microspheres are countedper sample. Control microspheres (paclitaxel absent) are prepared andsized as described previously.

B. Encapsulation Efficiency

Known weights of paclitaxel-loaded microspheres are dissolved in 1 mLDCM, 20 mL of 40% acetonitrile in water at 50° C. are added and vortexeduntil the DCM had been evaporated. The concentration of paclitaxel inthe 40% acetonitrile is determined by HPLC using a mobile phase ofwater:methanol:acetonitrile (37:5:58) at a flow rate of 1 mL/min(Beckman isocratic pump), a C8 reverse phase column (Beckman) and UVdetection at 232 nm. To determine the recovery efficiency of thisextraction procedure, known weights of paclitaxel from 100-1000 μg aredissolved in 1 mL of DCM and subjected to the same extraction procedurein triplicate as described previously. Recoveries are always greaterthan 85% and the values of encapsulation efficiency are correctedappropriately.

C. Drug Release Studies

In 15 mL glass, screw capped tubes are placed 10 mL of 10 mM phosphatebuffered saline (PBS), pH 7.4 and 35 mg paclitaxel-loaded microspheres.The tubes are tumbled at 37° C. and at given time intervals, centrifugedat 1500×g for 5 min and the supernatant saved for analysis. Microspherepellets are resuspended in fresh PBS (10 mL) at 37° C. and reincubated.Paclitaxel concentrations are determined by extraction into 1 mL DCMfollowed by evaporation to dryness under a stream of nitrogen,reconstitution in 1 mL of 40% acetonitrile in water and analysis usingHPLC as previously described.

D. Scanning Electron Microscopy (SEM)

Microspheres are placed on sample holders, sputter coated with gold andmicrographs obtained using a Philips 501B SEM operating at 15 kV.

E. CAM Studies

Fertilized, domestic chick embryos are incubated for 4 days prior toshell-less culturing. The egg contents are incubated at 90% relativehumidity and 3% CO₂ for 2 days. On day 6 of incubation, 1 mg aliquots of0.6% paclitaxel loaded or control (paclitaxel free) microspheres areplaced directly on the CAM surface. After a 2 day exposure thevasculature is examined using a stereomicroscope interfaced with a videocamera; the video signals are then displayed on a computer and videoprinted.

F. Results

Microspheres prepared from 100% EVA are freely suspended in solutions ofPVA but aggregated and coalesced or fused extensively on subsequentwashing in water to remove the PVA. Blending EVA with an increasingproportion of PLA produced microspheres showing a decreased tendency toaggregate and coalesce when washed in water, as described in FIG. 15A. A50:50 blend of EVA:PLA formed microspheres with good physical stability,that is the microspheres remained discrete and well suspended withnegligible aggregation and coalescence.

The size range for the “small” size fraction microspheres is determinedto be >95% of the microsphere sample (by weight) between 10-30 mm andfor the “large” size fraction, >95% of the sample (by weight) between30-100 mm. Representative scanning electron micrographs of paclitaxelloaded 50:50 EVA:PLA microspheres in the “small” and “large” size rangesare shown in FIGS. 15B and 15C, respectively. The microspheres arespherical with a smooth surface and with no evidence of solid drug onthe surface of the microspheres. The efficiency of loading 50:50 EVA:PLAmicrospheres with paclitaxel is between 95-100% at initial paclitaxelconcentrations of between 100-1000 mg paclitaxel per 50 mg polymer.There is no significant difference (Student t-test, p <0.05) between theencapsulation efficiencies for either “small” or “large” microspheres.

The time course of paclitaxel release from 0.6% w/v loaded 50:50 EVA:PLAmicrospheres is shown in FIG. 15D for “small” size (open circles) and“large” size (closed circles) microspheres. The release rate studies arecarried out in triplicate tubes in 3 separate experiments. The releaseprofiles are biphasic with an initial rapid release of paclitaxel or“burst” phase occurring over the first 4 days from both size rangemicrospheres. This is followed by a phase of much slower release. Thereis no significant difference between the release rates from “small” or“large” microspheres. Between 10-13% of the total paclitaxel content ofthe microspheres is released in 50 days.

The paclitaxel loaded microspheres (0.6% w/v loading) are tested usingthe CAM assay and the results are shown in FIG. 15E. The paclitaxelmicrospheres released sufficient drug to produce a zone of avascularityin the surrounding tissue (FIG. 15F). Note that immediately adjacent tothe microspheres (“MS” in FIGS. 15E and 15F) is an area in which bloodvessels are completely absent (Zone 1); further from the microspheres isan area of disrupted, non-functioning capillaries (Zone 2); it is onlyat a distance of approximately 6 mm from the microspheres that thecapillaries return to normal. In CAMs treated with control microspheres(paclitaxel absent) there is a normal capillary network architecture(figure not shown.)

Discussion

Arterial chemoembolization is an invasive surgical technique. Therefore,ideally, a chemoembolic formulation of an anti-angiogenic drug such aspaclitaxel would release the drug at the tumor site at concentrationssufficient for activity for a prolonged period of time, of the order ofseveral months. EVA is a tissue compatible nondegradable polymer whichhas been used extensively for the controlled delivery of macromoleculesover long time periods (>100 days).

EVA is initially selected as a polymeric biomaterial for preparingmicrospheres with paclitaxel dispersed in the polymer matrix. However,microspheres prepared with 100% EVA aggregated and coalesced a lnostcompletely during the washing procedure.

Polymers and copolymers based on lactic acid and glycolic acid arephysiologically inert and biocompatible and degrade by hydrolysis totoxicologically acceptable products. Copolymers of lactic acid andglycolic acids have faster degradation rates than PLA and drug loadedmicrospheres prepared using these copolymers are unsuitable forprolonged, controlled release over several months. Dollinger and Sawanblended PLA with EVA and showed that the degradation lifetime of PLA isincreased as the proportion of EVA in the blend is increased. Theysuggested that blends of EVA and PLA should provide a polymer matrixwith better mechanical stability and control of drug release rates thanPLA.

FIG. 15A shows that increasing the proportion of PLA in a EVA:PLA blenddecreased the extent of aggregation of the microsphere suspensions.Blends of 50% or less EVA in the EVA:PLA matrix produced physicallystable microsphere suspensions in water or PBS. A blend of 50:50 EVA:PLAis selected for all subsequent studies.

Different size range fractions of microspheres could be prepared bychanging the concentration of the emulsifier, PVA, in the aqueous phase.“Small” microspheres are produced at the higher PVA concentration of 5%w/v whereas “large” microspheres are produced at 2.5% w/v PVA. All otherproduction variables are the same for both microsphere size fractions.The higher concentration of emulsifier gave a more viscous aqueousdispersion medium and produced smaller droplets ofpolymer/paclitaxel/DCM emulsified in the aqueous phase and thus smallermicrospheres. The paclitaxel loaded microspheres contained between95-100% of the initial paclitaxel added to the organic phaseencapsulated within the solid microspheres. The low water solubility ofpaclitaxel favoured partitioning into the organic phase containing thepolymer.

Release rates of paclitaxel from the 50:50 EVA:PLA microspheres are veryslow with less than 15% of the loaded paclitaxel being released in 50days. The initial burst phase of drug release may be due to diffusion ofdrug from the superficial region of the microspheres (close to themicrosphere surface).

The mechanism of drug release from nondegradable polymeric matrices suchas EVA is thought to involve the diffusion of water through thedispersed drug phase within the polymer, dissolution of the drug anddiffusion of solute through a series of interconnecting, fluid filledpores. Blends of EVA and PLA have been shown to be immiscible orbicontinuous over a range of 30 to 70% EVA in PLA. In degradationstudies in PBS buffer at 37° C., following an induction or lag period,PLA hydrolytically degraded and eroded from the EVA:PLA polymer blendmatrix leaving an inactive sponge-like skeleton. Although the inductionperiod and rate of PLA degradation and erosion from the blended matricesdepended on the proportion of PLA in the matrix and on process history,there is consistently little or no loss of PLA until after 40-50 days.

Although some erosion of PLA from the 50:50 EVA:PLA microspheres mayhave occurred within the 50 days of the in vitro release rate study(FIG. 15C), it is likely that the primary mechanism of drug release fromthe polymer blend is diffusion of solute through a pore network in thepolymer matrix.

At the conclusion of the release rate study, the microspheres areanalyzed from the amount of drug remaining. The values for the percentof paclitaxel remaining in the 50 day incubation microsphere samples are94% ±9% and 89% ±12% for “large” and “small” size fraction microspheres,respectively.

Microspheres loaded with 6 mg per mg of polymer (0.6%) providedextensive inhibition of angiogenesis when placed on the CAM of theembryonic chick (FIGS. 15E and 15F).

Example 12 Paclitaxel Encapsulation in Poly(E-Caprolactone)Microspheres. Inhibition of Angiogenesis on the CAM Assay byPaclitaxel-Loaded Microspheres

This example evaluates the in vitro release rate profile of paclitaxelfrom biodegradable microspheres of poly(e-caprolactone) and demonstratesthe anti-angiogenic activity of paclitaxel released from thesemicrospheres when placed on the CAM.

Reagents which were utilized in these experiments include:poly(e-caprolactone) (“PCL”) (molecular weight 35,000-45,000; purchasedfrom Polysciences (Warrington, Pa.)); dichloromethane (“DCM”) fromFisher Scientific Co., Canada; polyvinyl alcohol (PVP) (molecular weight12,00-18,000, 99% hydrolysed) from Aldrich Chemical Co. (Milwaukee,Wis.), and paclitaxel from Sigma Chemical Co. (St. Louis, Mo.). Unlessotherwise stated all chemicals and reagents are used as supplied.Distilled water is used throughout.

A. Preparation of Microspheres

Microspheres are prepared essentially as described in Example 8utilizing the solvent evaporation method. Briefly, 5% w/w paclitaxelloaded microspheres are prepared by dissolving 10 mg of paclitaxel and190 mg of PCL in 2 ml of DCM, adding to 100 ml of 1% PVP aqueoussolution and stirring at 1000 rpm at 25° C. for 2 hours. The suspensionof microspheres is centrifuged at 1000×g for 10 minutes (Beckman GPR),the supernatant removed and the microspheres washed three times withwater. The washed microspheres are air-dried overnight and stored atroom temperature. Control microspheres (paclitaxel absent) are preparedas described above. Microspheres containing 1% and 2% paclitaxel arealso prepared. Microspheres are sized using an optical microscope with astage micrometer.

B. Encapsulation Efficiency

A known weight of drug-loaded microspheres (about 5 mg) is dissolved in8 ml of acetonitrile and 2 ml distilled water is added to precipitatethe polymer. The mixture is centrifuged at 1000 g for 10 minutes and theamount of paclitaxel encapsulated is calculated from the absorbance ofthe supernatant measured in a UV spectrophotometer (Hewlett-Packard8452A Diode Array Spectrophotometer) at 232 nm.

C. Drug Release Studies

About 10 mg of paclitaxel-loaded microspheres are suspended in 20 ml of10 mM phosphate buffered saline, pH 7.4 (PBS) in screw-capped tubes. Thetubes are tumbled end-over-end at 37° C. and at given time intervals19.5 ml of supernatant is removed (after allowing the microspheres tosettle at the bottom), filtered through a 0.45 um membrane filter andretained for paclitaxel. analysis. An equal volume of PBS is replaced ineach tube to maintain sink conditions throughout the study. Thefiltrates are extracted with 3×1 ml DCM, the DCM extracts evaporated todryness under a stream of nitrogen, redissolved in 1 ml acetonitrile andanalyzed by HPLC using a mobile phase of water:methanol:acetonitrile(37:5:58) at a flow rate of 1 ml min⁻¹ (Beckman Isocratic Pump), a C8reverse phase column (Beckman), and UV detection (Shimadzu SPD A) at 232nm.

D. CAM Studies

Fertilized, domestic chick embryos are incubated for 4 days prior toshell-less culturing. On day 6 of incubation, 1 mg aliquots of 5%paclitaxel-loaded or control (paclitaxel-free) microspheres are placeddirectly on the CAM surface. After a 2-day exposure the vasculature isexamined using a stereomicroscope interfaced with a video camera; thevideo signals are then displayed on a computer and video printed.

E. Scanning Electron Microscopy

Microspheres are placed on sample holders, sputter-coated with gold andthen placed in a Philips 501B Scanning Electron Microscope operating at15 kV.

F. Results

The size range for the microsphere samples is between 30-100 um,although there is evidence in all paclitaxel-loaded or controlmicrosphere batches of some microspheres falling outside this range. Theefficiency of loading PCL microspheres with paclitaxel is always greaterthan 95% for all drug loadings studied. Scanning electron microscopydemonstrated that the microspheres are all spherical and many showed arough or pitted surface morphology. There appeared to be no evidence ofsolid drug on the surface of the microspheres.

The time courses of paclitaxel release from 1%, 2% and 5% loaded PCLmicrospheres are shown in FIG. 16A. The release rate profiles arebi-phasic. There is an initial rapid release of paclitaxel or “burstphase” at all drug loadings. The burst phase occurred over 1-2 days at1% and 2% paclitaxel loading and over 3-4 days for 5% loadedmicrospheres. The initial phase of rapid release is followed by a phaseof significantly slower drug release. For microspheres containing 1% or2% paclitaxel there is no further drug release after 21 days. At 5%paclitaxel loading, the microspheres had released about 20% of the totaldrug content after 21 days.

FIG. 16B shows CAMs treated with control PCL microspheres, and FIG. 16Cshows treatment with 5% paclitaxel loaded microspheres. The CAM with thecontrol microspheres shows a normal capillary network architecture. TheCAM treated with paclitaxel-PCL microspheres shows marked vascularregression and zones which are devoid of a capillary network.

G. Discussion

The solvent evaporation method of manufacturing paclitaxel-loadedmicrospheres produced very high paclitaxel encapsulation efficiencies ofbetween 95-100%. This is due to the poor water solubility of paclitaxeland its hydrophobic nature favouring partitioning in the organic solventphase containing the polymer.

The biphasic release profile for paclitaxel is typical of the releasepattern for many drugs from biodegradable polymer matrices.Poly(e-caprolactone) is an aliphatic polyester which can be degraded byhydrolysis under physiological conditions and it is non-toxic and tissuecompatible. The degradation of PCL is significantly slower than that ofthe extensively investigated polymers and copolymers of lactic andglycolic acids and is therefore suitable for the design of long-termdrug delivery systems. The initial rapid or burst phase of paclitaxelrelease is thought to be due to diffusional release of the drug from thesuperficial region of the microspheres (close to the microspheresurface). Release of paclitaxel in the second (slower) phase of therelease profiles is not likely due to degradation or erosion of PCLbecause studies have shown that under in vitro conditions in water thereis no significant weight loss or surface erosion of PCL over a 7.5-weekperiod. The slower phase of paclitaxel release is probably due todissolution of the drug within fluid-filled pores in the polymer matrixand diffusion through the pores. The greater release rate at higherpaclitaxel loading is probably a result of a more extensive pore networkwithin the polymer matrix.

Paclitaxel microspheres with 5% loading have been shown to releasesufficient drug to produce extensive inhibition of angiogenesis whenplaced on the CAM. The inhibition of blood vessel growth resulted in anavascular zone as shown in FIG. 16C.

Example 13 Paclitaxel-Loaded Polymeric Films Composed of Ethlene VinylAcetate and a Surfactant

Two types of films are investigated within this example: pure EVA filmsloaded with paclitaxel and EVA/surfactant blend films loaded withpaclitaxel.

The surfactants being examined are two hydrophobic surfactants (Span 80and Pluronic L101) and one hydrophilic surfactant (Pluronic F127). Thepluronic surfactants are themselves polymers, which is an attractiveproperty since they can be blended with EVA to optimize various drugdelivery properties. Span 80 is a smaller molecule which is in somemanner dispersed in the polymer matrix, and does not form a blend.

Surfactants is useful in modulating the release rates of paclitaxel fromfilms and optimizing certain physical parameters of the films. Oneaspect of the surfactant blend films which indicates that drug releaserates can be controlled is the ability to vary the rate and extent towhich the compound will swell in water. Diffusion of water into apolymer-drug matrix is critical to the release of drug from the carrier.FIGS. 17C and 17D show the degree of swelling of the films as the levelof surfactant in the blend is altered. Pure EVA films do not swell toany significant extent in over 2 months. However, by increasing thelevel of surfactant added to the EVA it is possible to increase thedegree of swelling of the compound, and by increasing hydrophilicityswelling can also be increased.

Results of experiments with these films are shown below in FIGS. 17A-E.Briefly, FIG. 17A shows paclitaxel release (in mg) over time from pureEVA films. FIG. 17B shows the percentage of drug remaining for the samefilms. As can be seen from these two figures, as paclitaxel loadingincreases (i.e., percentage of paclitaxel by weight is increased), drugrelease rates increase, showing the expected concentration dependence.As paclitaxel loading is increased, the percent paclitaxel remaining inthe film also increases, indicating that higher loading may be moreattractive for long-term release formulations.

Physical strength and elasticity of the films is assessed in FIG. 17E.Briefly, FIG. 17E shows stress/strain curves for pure EVA andEVA-Surfactant blend films. This crude measurement of stressdemonstrates that the elasticity of films is increased with the additionof Pluronic F127, and that the tensile strength (stress on breaking) isincreased in a concentration dependent manner with the addition ofPluronic F127. Elasticity and strength are important considerations indesigning a film which can be manipulated for particular clinicalapplications without causing permanent deformation of the compound.

The above data demonstrates the ability of certain surfactant additivesto control drug release rates and to alter the physical characteristicsof the vehicle.

Example 14 Incorporating Methoxypolyethylene Glycol 350 (MePEG) intoPoly(E-Caprolactone) to Develop a Formulation for the ControlledDelivery of Paclitaxel from a Paste

Reagents and equipment which were utilized within these experimentsinclude methoxypolyethylene glycol 350 (“MePEG”—Union Carbide, Danbury,Conn.). MePEG is liquid at room temperature, and has a freezing point of10° to −5° C.

A. Preparation of a MePEG/PCL Paclitaxel-Containing Paste

MePEG/PCL paste is prepared by first dissolving a quantity of paclitaxelinto MePEG, and then incorporating this into melted PCL. One advantagewith this method is that no DCM is required.

B. Analysis of Melting Point

The melting point of PCL/MePEG polymer blends may be determined bydifferential scanning calorimetry from 30° C. to 70° C. at a heatingrate of 2.5° C. per minute. Results of this experiment are shown inFIGS. 18A and 18B. Briefly, as shown in FIG. 18A the melting point ofthe polymer blend (as determined by thermal analysis) is decreased byMePEG in a concentration dependent manner. The melting point of thepolymer blends as a function of MePEG concentration is shown in FIG.18A. This lower melting point also translates into an increased time forthe polymer blends to solidify from melt as shown in FIG. 18B. A 30:70blend of MePEG:PCL takes more than twice as long to solidify from thefluid melt than does PCL alone.

C. Measurement of Brittleness

Incorporation of MePEG into PCL appears to produce a less brittle solid,as compared to PCL alone. As a “rough” way of quantitating this, aweighted needle is dropped from an equal height into polymer blendscontaining from 0% to 30% MePEG in PCL, and the distance that the needlepenetrates into the solid is then measured. The resulting graph is shownas FIG. 18C. Points are given as the average of four measurements ±1S.D.

For purposes of comparison, a sample of paraffin wax is also tested andthe needle penetrated into this a distance of 7.25 mm±0.3 mm.

D. Measurement of Paclitaxel Release

Pellets of polymer (PCL containing 0%, 5%, 10% or 20% MePEG) areincubated in phosphate buffered saline (PBS, pH 7.4) at 37° C., and %change in polymer weight is measured over time. As can be seen in FIG.18D, the amount of weight lost increases with the concentration of MePEGoriginally present in the blend. It is likely that this weight loss isdue to the release of MePEG from the polymer matrix into the incubatingfluid. This would indicate that paclitaxel will readily be released froma MePEG/PCL blend since paclitaxel is first dissolved in MePEG beforeincorporation into PCL.

E. Effect of Varying Quantities of MePEG on Paclitaxel Release

Thermopastes are made up containing between 0.8% and 20% MePEG in PCL.These are loaded with 1% paclitaxel. The release of paclitaxel over timefrom 10 mg pellets in PBS buffer at 37° C. is monitored using HPLC. Asis shown in FIG. 18E, the amount of MePEG in the formulation does notaffect the amount of paclitaxel that is released.

F. Effect of Varying Quantities of Paclitaxel on the Total Amount ofPaclitaxel Released From a 20% MePEG/PCL Blend

Thermopastes are made up containing 20% MePEG in PCL and loaded withbetween 0.2% and 10% paclitaxel. The release of paclitaxel over time ismeasured as described above. As shown in FIG. 18F, the amount ofpaclitaxel released over time increases with increased paclitaxelloading. When plotted as the percent total paclitaxel released, however,the order is reversed (FIG. 18G). This gives information about theresidual paclitaxel remaining in the paste and allows for a projectionof the period of time over which paclitaxel may be released from the 20%MePEG Thermopaste.

G. Strength Analysis of Various MePEG/PCL Blends

A CT-40 mechanical strength tester is used to measure the strength ofsolid polymer “tablets” of diameter 0.88 cm and an average thickness of0.560 cm. The polymer tablets are blends of MePEG at concentrations of0%, 5%, 10% or 20% in PCL.

Results of this test are shown in FIG. 18H, where both the tensilestrength and the time to failure are plotted as a function of % MePEG inthe blend. Single variable ANOVA indicated that the tablet thicknesseswithin each group are not different. As can be seen from FIG. 18H, theaddition of MePEG into PCL decreased the hardness of the resultingsolid.

Example 15 Effect of Paclitaxel-Loaded Thermopaste on Angiogenesis InVivo

Fertilized, domestic chick embryos were incubated for 4 days prior toshell-less culturing as described in Example 2. The egg contents areremoved from the shell and emptied into roundbottom sterilized glassbowls and covered with petri dish covers.

Paclitaxel is incorporated into thermopaste at concentrations of 0.05%,0.1%, 0.25%, 0.5%, 1.0%, 5%, 10%, and 20% (w/v) essentially as describedabove (see Example 1.0), and used in the following experiments on theCAM. Dried thermopaste disks weighing 3 mg were made by heating thepaste to 60° C., forming drop size aliquots, and then allowing it tocool.

In addition, unloaded thermopaste and thermopaste containing 20%paclitaxel were also heated to 60° C. and placed directly on the growingedge of each CAM at day 6 of incubation; two animals each were treatedin this manner. There Was no observable difference in the resultsobtained using the different methods of administration indicating thatthe temperature of the paste at the time of application was not a factorin the outcome.

Each concentration of paclitaxel-loaded thermopaste (0.05%, 0.1%, 0.25%,0.5%, 1.0%, 5%, 10%, and 20%) was tested on 4 to 9 embryos at day 6 ofdevelopment (see Table III). After a 2 day exposure (day 8 ofincubation) the vasculature was examined with the aid of astereomicroscope. Liposyn II, a white opaque solution, was injected intothe CAM which increases the visibility of the vascular details.

The 20% paclitaxel-loaded thermopaste induced an extensive area ofavascularity (see FIG. 19B) in all 6 of the CAMs receiving thistreatment. The highest degree of inhibition was defined as a region ofavascularity covering an area of 6 mm in diameter. All of the CAMstreated with 20% paclitaxel-loaded thermopaste displayed this degree ofangiogenesis inhibition.

In the animals treated with 5% paclitaxel-loaded paste, 4 animalsdemonstrated maximum inhibition of angiogenesis. Of the animals treatedwith 10% paclitaxel-loaded thermopaste, only 5 illustrated maximalinhibition.

The results of this study also show that paclitaxel-loaded thermopaste,as low as 0.25%, can release a significant amount of drug to induceangiogenesis inhibition on the CAM. (Table IV, FIGS. 19C and 19D).

By comparison, the control (unloaded) thermopaste did not inhibitangiogenesis on the CAM (see FIG. 19A); this higher magnification view(note that the edge of the paste is seen at the top of the image)demonstrates that the vessels adjacent to the paste are unaffected bythe thermopaste. This suggests that the avascular effect observed is dueto the sustained release of paclitaxel and is not due to the polymeritself or due to a secondary pressure effect of the paste on thedeveloping vasculature.

This study also demonstrates that thermopaste releases sufficientquantities of angiogenesis inhibitor (in this case paclitaxel) toinhibit the normal development of the CAM vasculature. TABLE IVAngiogenic Inhibition of Paclitaxel-Loaded Thermopaste Paclitaxel-loadedEmbryos Evaluated Thermopaste (%) (positive/n)   0.05 0/9   0.1 1/8  0.25 4/4   0.5 4/4 1 8/8 5 4/4 10  5/5 20  6/6 0  0/30 (control)

Example 16 Effect of Paclitaxel-Loaded Thermopaste on Tumor Growth andTumor Angiogenesis In Vivo

Fertilized domestic chick embryos are incubated for 3 days prior tohaving their shells removed. The egg contents are emptied by removingthe shell located around the airspace, severing the interior shellmembrane, perforating the opposite end of the shell and allowing the eggcontents to gently slide out from the blunted end. The contents areemptied into round-bottom sterilized glass bowls, covered with petridish covers and incubated at 90% relative humidity and 3% carbon dioxide(see Example 2).

MDAY-D2 cells (a murine lymphoid tumor) is injected into mice andallowed to grow into tumors weighing 0.5-1.0 g. The mice are sacrificed,the tumor sites wiped with alcohol, excised, placed in sterile tissueculture media, and diced into 1 mm pieces under a laminar flow hood.Prior to placing the dissected tumors onto the 9-day old chick embryos,CAM surfaces are gently scraped with a 30 gauge needle to insure tumorimplantation. The tumors are then placed on the CAMs after 8 days ofincubation (4 days after desbelling), and allowed to grow on the CAM forfour days to establish a vascular supply. Four embryos are preparedutilizing this method, each embryo receiving 3 tumors. For theseembryos, one tumor receives 20% paclitaxel-loaded thermopaste, thesecond tumor unloaded thermopaste, and the third tumor no treatment. Thetreatments are continued for two days before the results were recorded.

The explanted MDAY-D2 tumors secrete angiogenic factors which induce theingrowth of capillaries (derived from the CAM) into the tumor mass andallow it to continue to grow in size. Since all the vessels of the tumorare derived from the CAM, while all the tumor cells are derived from theexplant, it is possible to assess the effect of therapeuticinterventions on these two processes independently. This assay has beenused to determine the effectiveness of paclitaxel-loaded thermopaste on:(a) inhibiting the vascularization of the tumor and (b) inhibiting thegrowth of the tumor cells themselves.

Direct in vivo stereomicroscopic evaluation and histological examinationof fixed tissues from this study demonstrated the following. In thetumors treated with 20% paclitaxel-loaded thermopaste, there was areduction in the number of the blood vessels which supplied the tumor(see FIGS. 20C and 20D), a reduction in the number of blood vesselswithin the tumor, and a reduction in the number of blood vessels in theperiphery of the tumor (the area which is typically the most highlyvascularized in a solid tumor) when compared to control tumors. Thetumors began to decrease in size and mass during the two days the studywas conducted. Additionally, numerous endothelial cells were seen to bearrested in cell division indicating that endothelial cell proliferationhad been affected. Tumor cells were also frequently seen arrested inmitosis. All 4 embryos showed a consistent pattern with the 20%paclitaxel-loaded thermopaste suppressing tumor vascularity while theunloaded thermopaste had no effect.

By comparison, in CAMs treated with unloaded thermopaste, the tumorswere well vascularized with an increase in the number and density ofvessels when compared to that of the normal surrounding tissue, anddramatically more vessels than were observed in the tumors treated withpaclitaxel-loaded paste. The newly formed vessels entered the tumor fromall angles appearing like spokes attached to the center of a wheel (seeFIGS. 20A and 20B). The control tumors continued to increase in size andmass during the course of the study. Histologically, numerous dilatedthin-walled capillaries were seen in the periphery of the tumor and fewendothelial were seen to be in cell division. The tumor tissue was wellvascularized and viable throughout.

As an example, in two similarly-sized (initially, at the time ofexplantation) tumors placed on the same CAM the following data wasobtained. For the tumor treated with 20% paclitaxel-loaded thermopastethe tumor measured 330 mm×597 mm; the immediate periphery of the tumorhas 14 blood vessels, while the tumor mass has only 3-4 smallcapillaries. For the tumor treated with unloaded thermopaste the tumorsize was 623 mm×678 mm; the immediate periphery of the tumor has 54blood vessels, while the tumor mass has 12-14 small blood vessels. Inaddition, the surrounding CAM itself contained many more blood vesselsas compared to the area surrounding the paclitaxel-treated tumor.

This study demonstrates that thermopaste releases sufficient quantitiesof angiogenesis inhibitor (in this case paclitaxel) to inhibit thepathological angiogenesis which accompanies tumor growth anddevelopment. Under these conditions angiogenesis is maximally stimulatedby the tumor cells which produce angiogenic factors capable of inducingthe ingrowth of capillaries from the surrounding tissue into the tumormass. The 20% paclitaxel-loaded thermopaste is capable of blocking thisprocess and limiting the ability of the tumor tissue to maintain anadequate blood supply. This results in a decrease in the tumor mass boththrough a cytotoxic effect of the drug on the tumor cells themselves andby depriving the tissue of the nutrients required for growth andexpansion.

Example 17 Effect of Angiogenesis Inhibitor-Loaded Thermopaste on TumorGrowth In Vivo in a Murine Tumor Model

The murine MDAY-D2 tumor model may be used to examine the effect oflocal slow release of the chemotherapeutic and anti-angiogenic compoundssuch as paclitaxel on tumor growth, tumor metastasis, and animalsurvival. The MDAY-D2 tumor cell line is grown in a cell suspensionconsisting of 5% Fetal Calf Serum in alpha mem media. The cells areincubated at 37° C. in a humidified atmosphere supplemented with 5%carbon dioxide, and are diluted by a factor of 15 every 3 days until asufficient number of cells are obtained. Following the incubation periodthe cells are examined by light microscopy for viability and then arecentrifuged at 1500 rpm for 5 minutes. PBS is added to the cells toachieve a dilution of 1,000,000 cells per ml.

Ten week old DBA/2j female mice are acclimatized for 3-4 days afterarrival. Each mouse is then injected subcutaneously in theposteriolateral flank with 100,000 MDAY-D2 cells in 100 ml of PBS.Previous studies have shown that this procedure produces a visible tumorat the injection site in 3-4 days, reach a size of 1.0-1.7 g by 14 days,and produces visible metastases in the liver 19-25 days post-injection.Depending upon the objective of the study a therapeutic intervention canbe instituted at any point in the progression of the disease.

Using the above animal model, 20 mice are injected with 140,000 MDAY-D2cells s.c. and the tumors allowed to grow. On day 5 the mice are dividedinto groups of 5. The tumor site was surgically opened under anesthesia,the local region treated with the drug-loaded thermopaste or controlthermopaste without disturbing the existing tumor tissue, and the woundwas closed. The groups of 5 received either no treatment (wound merelyclosed), polymer (PCL) alone, 10% paclitaxel-loaded thermopaste, or 20%paclitaxel-loaded thermopaste (only 4 animals injected) implantedadjacent to the tumor site. On day 16, the mice were sacrificed, thetumors were dissected and examined (grossly and histologically) fortumor growth, tumor metastasis, local and systemic toxicity resultingfrom the treatment, effect on wound healing, effect on tumorvascularity, and condition of the paste remaining at the incision site.

The weights of the tumors for each animal is shown in the table below:TABLE V Tumor Weights (gm) Control Control 10% Paclitaxel 20% PaclitaxelAnimal No. (empty) (PCL) Thermopaste Thermopaste 1 1.387 1.137 0.4870.114 2 0.589 0.763 0.589 0.192 3 0.461 0.525 0.447 0.071 4 0.606 0.2820.274 0.042 5 0.353 0.277 0.362 Mean 0.6808 0.6040 0.4318 0.1048 Std.Deviation 0.4078 0.3761 0.1202 0.0653 P Value 0.7647 0.358 0.036Thermopaste loaded with 20% paclitaxel reduced tumor growth by over 85%(average weight 0.105) as compared to control animals (average weight0.681). Animals treated with thermopaste alone or thermopaste containing10% paclitaxel had only modest effects on tumor growth; tumor weightswere reduced by only 10% and 35% respectively (FIG. 21A). Therefore,thermopaste containing 20% paclitaxel was more effective in reducingtumor growth than thermopaste containing 10% paclitaxel (see FIG. 21C;see also FIG. 21B).

Thermopaste was detected in some of the animals at the site ofadministration. Polymer varying in weight between 0.026 g to 0.078 g wasdetected in 8 of 15 mice. Every animal in the group containing 20%paclitaxel-loaded thermopaste contained some residual polymer suggestingthat it was less susceptible to dissolution. Histologically, the tumorstreated with paclitaxel-loaded thermopaste contained lower cellularityand more tissue necrosis than control tumors. The vasculature wasreduced and endothelial cells were frequently seen to be arrested incell division. The paclitaxel-loaded thermopaste did not appear toaffect the integrity or cellularity of the skin or tissues surroundingthe tumor. Grossly, wound healing was unaffected.

Example 18 The use of Angiogenesis-Inhibitor Loaded Surgical Films inthe Prevention of Iatrogenic Metastatic Seeding of Tumor Cells DuringCancer Resection Surgery

As discussed above, sterile, pliable, stretchable drug-polymer compounds(e.g., films) may be utilized in accordance with the methods describedherein in order to isolate normal surrounding tissues from malignanttissue during resection of cancer. Such material prevents iatrogenicspread of the disease to adjacent organs through inadvertentcontamination by cancer cells. Such polymers may be particularly usefulif placed around the liver and/or other abdominal contents during bowelcancer resection surgery in order to prevent intraperitoneal spread ofthe disease to the liver.

A. Materials and Methods

Preparation of Surgical Film. Surgical films are prepared as describedin Example 10. Thin films measuring approximately 1 cm×1 cm are preparedcontaining either polymer alone (PCL) or PCL loaded with 5% paclitaxel.

Rat Hepatic Tumor Model. In an initial study Wistar rats weighingapproximately 300 g underwent general anesthesia and a 3-5 cm abdominalincision is made along the midline. In the largest hepatic lobe, a 1 cmincision is made in the hepatic parenchyma and part of the liver edge isresected. A concentration of 1 million live 9 L Glioma tumor cells(eluted from tissue culture immediately prior to the procedure)suspended in 100 ml of phosphate buffered saline are deposited onto thecut liver edge with a 30 gauge needle. The surgical is then placed overthe cut liver edge containing the tumor cells and affixed in place withGelfoam. Two animals received PCL films containing 5% paclitaxel and twoanimals received films containing PCL alone. The abdominal wall isclosed with 3.0 Dexon and skin clips. The general anesthetic isterminated and the animal is allowed to recover. Ten days later theanimals are sacrificed and the livers examined histologically.

B. Results

Local tumour growth is seen in the 2 livers treated with polymer alone.Both livers treated with polymer plus paclitaxel are completely free oftumour when examined histologically. Also of importance, the livercapsule had regenerated and grown completely over the polymeric film andthe cut surface of the liver indicating that there is no significanteffect on wound healing. There is no evidence of local hepatic toxicitysurrounding any (drug-loaded or drug-free) of the surgical films.

C. Discussion

This study indicates that surgical films placed around normal tissuesand incision sites during surgery may be capable of decreasing theincidence of accidental implantation of tumor cells into normalsurrounding tissue during resection of malignant tumors.

Example 19 Intra-Articular Injection of Angiogenesis-Inhibitor-LoadedBiodegradabel Microspheres in the Treatment of Arthritis

Articular damage in arthritis is due to a combination of inflammation(including WBCs and WBC products) and pannus tissue development (atissue composed on neovascular tissue, connective tissue, andinflammatory cells). Paclitaxel has been chosen for the initial studiesbecause it is a potent inhibitor of neovascularization. In this manner,paclitaxel in high local concentrations will prove to be a diseasemodifying agent in arthritis.

In order to determine whether microspheres have a deleterious effect onjoints, the following experiments are conducted. Briefly, plain PCL andpaclitaxel-loaded microspheres are prepared as described previously inExample 8. Three rabbits are injected intra-articularly with 0.5-5.0 μm,10-30 μm, or 30-80 μm microspheres in a total volume of 0.2 mls(containing 0.5 mg of microspheres). The joints are assessed visually(clinically) on a daily basis. After two weeks the animals aresacrificed and the joints examined histologically for evidence ofinflammation and depletion of proteoglycans.

The rabbit inflammatory arthritis and osteoarthritis models may beutilized in order to evaluate the use of microspheres in reducingsynovitis and cartilage degradation. Briefly, degenerative arthritis isinduced by a partial tear of the cruciate ligament and meniscus of theknee. After 4 to 6 weeks, the rabbits develop erosions in the cartilagesimilar to that observed in human osteoarthritis. Inflammatory arthritisis induced by immunizing rabbits with bovine serum albumen (BSA) inComplete Freund's Adjuvant (CFA). After 3 weeks, rabbits containing ahigh titer of anti-BSA antibody receive an intra-articular injection ofBSA (5 mg). Joint swelling and pronounced synovitis is apparent by sevendays, a proteoglycan depletion is observed by 7 to 14 days, andcartilage erosions are observed by 4 to 6 weeks.

Inflammatory arthritis is induced as described above. After 4 days, thejoints are injected with microspheres containing 5% paclitaxel orvehicle. One group of animals is sacrificed on day 14 and another on day28. The joints are examined histologically for inflammation andcartilage degradation. The experiment is designed to determine ifpaclitaxel microspheres can affect joint inflammation and cartilagematrix degradation.

Angiogenesis-inhibitor microspheres may be further examined in anosteoarthritis model. Briefly, degenerative arthritis is induced inrabbits as described above, and the joints receive an intra-articularinjection of microspheres (5% paclitaxel or vehicle only) on day 4. Theanimals are sacrificed on day 21 and day 42 and the joints examinedhistologically for evidence of cartilage degradation.

Results

Unloaded PCL microspheres of differing sizes (0.5-5.0 μm, 10-30 μm, or30-80 μm) are injected intra-articularly into the rabbit knee joint.Results of these experiments are shown in FIGS. 22A to 22D. Briefly,FIG. 22A is a photograph of synovium from PBS injected joints. FIG. 22Bis a photograph of joints injected with microspheres. FIG. 22C is aphotograph of cartilage from joints injected with PBS, and FIG. 22D is aphotograph of cartilage from joints injected with microspheres.

As can be seen from these photographs, histologically, there is nodifference between joints receiving a microsphere injection and thosewhich did not. Clinically, there was no evidence of joint inflammationduring the 14 days the experiment was conducted. Grossly, there is noevidence of joint inflammation or cartilage damage in joints wheremicrospheres are injected, as compared to untreated normal joints.

Conclusions

Microspheres can be injected intra-articularly without causing anydiscernible changes to the joint surface. This indicates that thismethod may be an effective means of delivering a targeted,sustained-release of disease-modifying agents to diseased joints, whileminimizing the toxicity which could be associated with the systemicadministration of such biologically active compounds.

As discussed above, microspheres can be formulated into specific sizeswith defined drug release kinetics. It has also been demonstrated thatpaclitaxel is a potent inhibitor of angiogenesis and that it is releasedfrom microspheres in quantities sufficient to block neovascularizationon the CAM assay. Therefore, angiogenesis-inhibitor-loaded (e.g.,paclitaxel-loaded) microspheres may be administered intra-articularly inorder to block the neovascularization that occurs in diseases such asrheumatoid arthritis. In this manner the drug-loaded microspheres canact as a “chondroprotective” agent which protects the cartilage fromirreversible destruction from invading neovascular pannus tissue.

Example 20 The Anti-Angiogenic Effects of Paclitaxel in an OphthalmicSuspension

In order to test whether paclitaxel would inhibit the pathogenesis ofcorneal neovascularization, an ophthalmic suspension of 0.3% paclitaxeland a 10% paclitaxel microsphere suspension was first prepared andtested on the CAM in order to determine whether sufficient quantities ofpaclitaxel could be released to inhibit angiogenesis.

Briefly, fertilized, chick eggs were incubated for 4 days prior toshell-less culturing as described previously in Example 2. The eggcontents are removed from the shell and emptied into round-bottomsterilized glass bowls and covered with petri dish covers.

On day 6 of development, the ophthalmic drops were tested on the CAM. Todeliver the ophthalmic suspensions, a 0.5 mL syringe was sliced intorings measuring 1 mm thick. These rings, which formed wells when placedonto the CAM were used to localize a 15 μL aliquot of ophthalmicsuspension to the CAM's blood vessels. The paclitaxel (0.3%) suspensionwas tested on 11 embryos, whereas the 10% paclitaxel-loaded microspheresuspension was tested on 4 other embryos. The control (unloaded)ophthalmic suspension was tested on the remaining 5 CAMs. After a 48hour period, Liposyn II, a white opaque solution, was injected into theCAM which increased the visibility of the vascular details when observedwith a stereomicroscope.

Within 48 hours, the 0.3% paclitaxel suspension inhibited angiogenesison 11/11 CAMs tested and the 10% paclitaxel-loaded microspheresuspension inhibited angiogenesis on 4/4 of the embryos tested. This wasevident by the presence of avascular zones measuring 6 mm in diameter inthe vicinity of the treated area (FIG. 23A); in many cases the avascularzone exceeded the size of the application ring. This avascular zone wasdefined as a region containing disrupted blood vessel fragments anddiscontinuous blood flow. The functional vessels adjacent to theavascular zone were modified in such a way to redirect the blood flowaway from the drug source; these vessels possessed an angulararchitecture which was not evident in the control (unloaded) thermopastetreated CAMs.

In comparison, the control (unloaded) ophthalmic vehicle did not inhibitangiogenesis on any of the 5 CAMs tested; this was evident by thefunctional vessels visible within the center of the application ring(FIG. 23B). In some cases, there was some reduction in the amount ofmicrovessels located in the control treated CAMs although this was onlydue to the aqueous suspension vehicle in which paclitaxel wasadministered.

In summary, paclitaxel was sufficiently released from the ophthalmicdrop suspension to inhibit angiogenesis on the CAM. Therefore, sincepaclitaxel can be released from this vehicle system, it may likewise beutilized in the treatment of neovascular disease of the eye, such ascorneal neovascularization.

Example 21 The Anti-Angiogenic Effects of Paclitaxel-Loaded StentCoating

A. Testing of Paclitaxel-Loaded Stents on a CAM

As noted above, stents are currently used for the prevention of luminalclosure induced by a disease process, such as biliary tumor ingrowth.Although stents prevent tumor ingrowth temporarily, tumor ingrowtheventually recurs. In this study, strecker stents were coated with anEVA polymer containing paclitaxel at concentrations of 33%, 10%, and2.5% and were tested for their ability to inhibit angiogenesis on theCAM.

Briefly, paclitaxel-coated stent tynes (3 mm in size) were placed ontothe growing vessels of the CAM at day 6 of development. Of these CAMs, 3received 33% paclitaxel-loaded stent coating, 5 CAMs received 2.5%paclitaxel, and 1 CAM received 10% paclitaxel-loaded stent coating. Inaddition, control stents, coated with unloaded EVA, were tested on atotal of 6 CAMs. After 48 hours, Liposyn II was injected within the CAMto increase the vascular details during observations.

All 3 different paclitaxel concentrations of the stent coating inhibitedangiogenesis on the CAM within the 48 hour period. The CAMs weremaximally inhibited which was characterized by the induction ofavascular zones measuring 6 mm in diameter (FIG. 24A).

B. Testing of Control Stents in Tumors

Similar to above, control stents were prepared as described above andplaced into established gliosarcoma tumors of the rat liver. After a 7day period, these rats were sedated and perfused with a 2.0%glutaraldehyde in sodium cacodylate solution. The livers were excisedand the stents were dissected away from the surrounding tissue. Imagesof the gross anatomy revealed that the nylon stents had becomeincorporated into the tumor and tumor ingrowth had been establishedwithin the lumen of the stent (FIGS. 25 and 26). FIG. 27 shows thatmetastasis had occurred within the lung.

Unlike the paclitaxel-loaded stent coating, the control coating did notinhibit angiogenesis and maintained its normal architecture in all ofthe 6 CAMs which were tested (FIG. 24B).

In summary, since paclitaxel coated stents have the capability ofreleasing sufficient drug to inhibit angiogenesis on the CAM, paclitaxelcoated stents may likewise be utilized for a variety of applications inorder to prevent tumor ingrowth within the binary lumen.

Example 22 Effect of Paclitaxel on Neutrophil Activity

The example describes the effect of paclitaxel on the response ofneutrophils stimulated with opsonized CPPD crystals or opsonizedzymosan. As shown by experiments set forth below, paclitaxel is a stronginhibitor of particulate inducted neutrophil activation as measured bychemiluminescence, superoxide anion production and degranulation inresponse to plasma opsonized microcrystals or zymosan.

A. Materials and Methods

Hanks buffered salt solution (HBSS) pH 7.4was used throughout thisstudy. All chemicals were purchased from Sigma Chemical Co (St. Louis,)unless otherwise stated. All experiments were performed at 37° C. unlessotherwise stated.

1. Preparation and Characterization of Crystals

CPPD (triclinic) crystals were prepared. The size distribution of thecrystals was approximately 33% less than 10 μm, 58% between 10 and 20 μmand 9% greater than 20 μm. Crystals prepared under the above conditionsare pyrogen free and crystals produced under sterile, pyrogen freeconditions produced the same magnitude of neutrophil response ascrystals prepared under normal, non-sterile laboratory conditions.

2. Opsonization of Crystals and Zymosan

All experiments that studied neutrophil responses to crystals or zymosanin the presence of paclitaxel were performed using plasma opsonized CPPDor zymosan. Opsonization of crystals or zymosan was done with 50%heparinized plasma at a concentration of 75 mg of CPPD or 12 mg ofzymosan per ml of 50% plasma. Crystals or zymosan were incubated withplasma for 30 min. at 37° C. and then washed in excess HBSS.

3. Neutrophil Preparation

Neutrophils were prepared from freshly collected human citrated wholeblood. Briefly, 400 ml of blood were mixed with 80 ml of 4% dextran T500(Phamacia LKB, Biotechnology AB Uppsala, Sweden) in HBSS and allowed tosettle for 1 h. Plasma was collected continuously and 5 ml applied to 5ml of Ficoll Paque (Pharmacia) in 15 ml polypropylene tubes (Corning,N.Y.). Following centrifugation at 500×g for. 30 min, the neutrophilpellets were washed free of erythrocytes by 20 s of hypotonic shock.Neutrophils were resuspended in HBSS, kept on ice and used forexperiments within 3 h. Neutrophil viability and purity was alwaysgreater than 90%.

4. Incubation of Neutrophils with Paclitaxel

A stock solution of paclitaxel at 12 mM in DMSO was freshly preparedbefore each experiment. This stock solution was diluted in DMSO to givesolutions of paclitaxel in the 1 to 10 mM concentration range. Equalvolumes of these diluted paclitaxel solutions was added to neutrophilsat 5,000,000 cells per ml under mild vortexing to achieve concentrationsof 0 to 50 μM with a final DMSO concentration of 0.5%. Cells wereincubated for 20 minutes at 33° C. then for 10 minutes at 37° C. beforeaddition to crystals or zymosan.

5. Chemiluminescence Assay

All chemiluminescence studies were performed at a cell concentration of5,000,000 cells/ml in HBSS with CPPD (50 mg/ml). In all experiments 0.5ml of cells was added to 25 mg of CPPD or 0.5 mg of zymosan in 1.5 mlcapped Eppendorf tubes. 10 μl of luminol dissolved in 25% DMSO in HBSSwas added to a final concentration of 1 μM and the samples were mixed toinitiate neutrophil activation by the crystals or zymosan.Chemiluminescence was monitored using an LKB Luminometer (Model 1250.)at 37° C. with shaking immediately prior to measurements to resuspendthe crystals or zymosan. Control tubes contained cells, drug and luminol(crystals absent).

6. Superoxide Anion Generation

Superoxide anion concentrations were measured using the superoxidedismutase inhibitable reduction of cytochrome c assay. Briefly, 25 mg ofcrystals or 0.5 mg of zymosan was placed in a 1.5 ml capped Eppendorftube and warmed to 37° C. 0.5 ml of cells at 370 C were added togetherwith ferricytochrome c (final concentration 1.2 mg/ml) and the cellswere activated by shaking the capped tubes. At appropriate times tubeswere centrifuged at 10,000 g for 10 seconds and the supernatantcollected for assay be measuring the absorbance of 550 nm. Control tubeswere set up under the same conditions with the inclusion of superoxidedismutase at 600 units per ml.

7. Neutrophil Degranulation Assay

One and a half milliliter Eppendorf tubes containing either 25 mg ofCPPD or 1 mg of zymosan were preheated to 37° C. 0.5 ml of cells at 37°C. were added followed by vigorous shaking to initiate the reactions. Atappropriate times, tubes were centrifuged at 10,000×g for 10 seconds and0.4 ml of supernatant was stored at −20° C. for later assay.

Lysozyme was measured by the decrease in absorbance at 450 nm of aMicrococcus lysodeikticus suspension. Briefly, Micrococcus lysodeikticuswas suspended at 0.1 mg/ml in 65 mM potassium phosphate buffer, pH 6.2and the absorbance at 450 nm was adjusted to 0.7 units by dilution. Thecrystal (or zymosan) and cell supernatant (100 μl) was added to 2.5 mlof the Micrococcus suspension and the decrease in absorbance wasmonitored. Lysozyme standards (chicken egg white) in the 0 to 2000units/ml range were prepared and a calibration graph of lyzozymeconcentration against the rate of decrease in the absorbance at 450 n mwas obtained.

Myeloperoxidase (MPO) activity was measured by the increase inabsorbance at 450 nm that accompanies the oxidation of dianisidine. 7.8mg of dianisidine was dissolved in 100 ml of 0.1 M citrate buffer, pH5.5 at 3.2 mM by sonication. To a 1 ml cuvette, 0.89 mL of thedianisidine solution was added, followed by 50 μl of 1% Triton×100, 10μl of a 0.05% hydrogen peroxide in water solution and 50 ul ofcrystal-cell supernatant. MPO activity was determined from the change inabsorbance (450 nm) per minute, Delta A 450, using the followingequation:Dianisidine oxidation (nmol/min)=50×Delta A 450

8. Neutrophil Viability

To determine the effect of paclitaxel on neutrophil viability therelease of the cytoplasmic marker enzyme, Lactate Dehydrogenase (LDH)was measured. Control tubes containing cells with drug (crystals absent)from degranulation experiments were also assayed for LDH.

B. Results

In all experiments statistical significance was determined usingStudents' t-test and significance was claimed at p<0.05. Where errorbars are shown they describe one standard deviation about the mean valuefor the n number given.

1. Neutrophil Viability

Neutrophils treated with paclitaxel at 46 μM for one hour at 37° C. didnot show any increased level of LDH release (always less than 5% oftotal) above controls indicating that paclitaxel did not cause celldeath.

2. Chemiluminescence

Paclitaxel at 28 μM produced strong inhibition of both plasma opsonizedCPPD and plasma opsonised zymosan induced neutrophil chemiluminescenceas shown in FIGS. 29A, 29B and 33A respectively. The inhibition of thepeak chemiluminescence response was 52% (±12%) and 45% (±11%) for CPPDand zymosan respectively. The inhibition by paclitaxel at 28 μM of bothplasma opsonized CPPD and plasma opsonized zymosan inducedchemiluminescence was significant at all times from 3 to 16 minutes(FIGS. 29A, 29B and 33A). FIGS. 29A and 29B show the concentrationdependence of paclitaxel inhibition of plasma opsonized CPPD inducedneutrophil chemiluminescence. In all experiments control samples neverproduced chemiluminescence values of greater than 5 mV and the additionof paclitaxel at all concentrations used in this study had no effect onthe chemiluminescence values of controls.

3. Superoxide Generation

The time course of plasma opsonised CPPD crystal induced superoxideanion production, as measured by the superoxide dismutase (SOD)inhibitable reduction of cytochrome c, is shown in FIG. 2. Treatment ofthe cells with paclitaxel at 28 μM produced a decrease in the amount ofsuperoxide generated at all times. This decrease was significant at alltimes shown in FIG. 30A. The concentration dependence of this inhibitionis shown in FIG. 30B. Stimulation of superoxide anion production byopsonised zymosan (FIG. 31B) showed a similar time course to CPPDinduced activation. The inhibition of zymosan induced superoxide anionproduction by paclitaxel at 28 μM was less dramatic than the inhibitionof CPPD activation but was significant at all times shown in FIG. 31B.

4. Neutrophil Degranulation

Neutrophil degranulation was monitored by the plasma opsonized CPPDcrystal induced release of myeloperoxidase and lysozyme or the plasmaopsonized zymosan induced release of myeloperoxidase. It has been shownthat sufficient amounts of these two enzymes are released into theextracellular media when plasma coated CPPD crystals are used tostimulate neutrophils without the need for the addition of cytochalasinB to the cells. FIGS. 32 and 33 show the time course of the release ofMPO and lysozyme respectively, from neutrophils stimulated by plasmacoated CPPD. FIG. 32A shows that paclitaxel inhibits myeloperoxidaserelease from plasma opsonized CPPD activated neutrophils in the first 9minutes of the crystal-cell incubation. Paclitaxel significantlyinhibited CPPD induced myeloperoxidase release at all times as shown inFIG. 32A. FIG. 32B shows the concentration dependence of paclitaxelinhibition of CPPD induced myeloperoxidase release.

Paclitaxel at 28 μM reduced lysozyme release and this inhibition ofdegranulation was significant at all times as shown in FIG. 33.

Only minor amounts of MPO and lysozyme were released when neutrophilswere stimulated with opsonized zymosan. Despite these low levels it waspossible to monitor 50% inhibition of MPO release after 9 ninutesincubation in the presence of paclitaxel at 28 μM that was statisticallysignificant (p<0.05) (data not shown).

C. Discussion

These experiments demonstrate that paclitaxel is a strong inhibitor ofcrystal induced neutrophil activation. In addition, by showing similarlevels of inhibition in neutrophil responses to another form ofparticulate activator, opsonized zymozan, it is evident that theinhibitory activity of paclitaxel is not limited to neutrophil responsesto crystals.

Example 23 Effect of Paclitaxel on Synoviocyte Proliferation

Two experiments were conducted in order to assess the effect ofdiffering concentrations of paclitaxel on tritiated thymidineincorporation (a measurement of synoviocyte DNA synthesis) andsynoviocyte proliferation in vitro.

A. Materials and Methods

1. ³H-Thymidine Incorporation into Synoviocytes

Synoviocytes were incubated with different concentrations of paclitaxel(10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M) continuously for 6 or 24 hours invitro. At these times, 1×10⁻⁶ cpm of ³H-thymidine was added to the cellculture and incubated for 2 hours at 37° C. The cells were placedthrough a cell harvester, washed through a filter, the filters were cut,and the amount of radiation contained in the filter sections determined.Once the amount of thymidine incorporated into the cells wasascertained, it was used to determine the rate of cell proliferation.This experiment was repeated three times and the data collated together.

2. Synoviocyte Proliferation

Bovine synovial fibroblasts were grown in the presence and absence ofdiffering concentrations (10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M) ofpaclitaxel for 24 hours. At the end of this time period the total numberof viable synoviocyte cells was determined visually by dye exclusioncounting using Trypan blue staining. This experiment was conducted 4times and the data collated.

B. Results

1. ³H-Thymidine Incorporation into Synoviocytes

This study demonstrated that paclitaxel at low concentrations inhibitsthe incorporation of 3H-thymidine (and by extension DNA synthesis) insynoviocytes at concentrations as low as 10⁻⁸ M. At six hours there wasno significant difference in the between the degree of inhibitionproduced by the higher versus the lower concentrations of paclitaxel(see FIG. 34). However, by 24 hours some of the effect was lost at lowerconcentrations of the drug (10⁻⁸ M), but was still substantially lowerthan that seen in control animals.

2. Synoviocyte Proliferation

This study demonstrated that paclitaxel was cytotoxic to proliferatingsynovial fibroblasts in a concentration dependent manner. Paclitaxel atconcentrations as low as 10⁻⁷ M is capable of inhibiting proliferationof the synoviocytes (see FIG. 35). At higher concentrations ofpaclitaxel (10⁻⁶ M and 10⁻⁵ M) the drug was toxic to the synovialfibroblasts in vitro.

C. Discussion

The above study demonstrates that paclitaxel is capable of inhibitingthe proliferation of synovial fibroblasts at relatively lowconcentrations in vitro. Therefore, given the role of these cells in thedevelopment of pannus tissue and their growth during the pathogenesis ofrheumatoid arthritis, blocking synoviocyte proliferation can be expectedto favorably affect the outcome of the disease in vivo.

Example 24 Effect of Paclitaxel on Collagenase Expression

As noted above, collagenase production by a variety of tissues (synovialfibroblasts, endothelial cells, chondrocytes, and white blood cells)plays an critical role in the development of the pathology of arthritis.Degradation of the cartilage matrix by proteolytic enzymes represents anirreversible step in the development of the disease resulting inirreparable damage to the articular cartilage. Numerous attempts havebeen made to restore the balance between the enzymes which degradeconnective tissue (matrix metalloproteinases—MMPs; collagenase is animportant member of this family) and those which inhibit degradation(tissue inhibitors of metalloproteinases—TIMPs). Evidence suggests thatthe imbalance of proteolytic versus inhibitory activity which results incartilage destruction is due to an excess of MMP activity as opposed toa paucity of TIMP activity. Treatment that decreases the amount of MMPactivity may thus favorably influence the outcome of the disease.

C-fos is an oncogene transcription factor shown to be involved andrequired for the induction of genes involved in cell proliferation andcollagenase expression. In cultured chondrocytes, both interleukin-1(IL-1) and tumor necrosis factor (TNF) have been shown to stimulatec-fos expression and produce all of the signals necessary to induce theexpression of collagenase. When IL-1 is administered to chondrocytes invitro there is a transient increase in fos mRNA levels which peak 30-60minutes later, while collagenase mRNA is detected 9 hours later andcontinues to increase up to 12 hours (data not shown) after IL-1stimulation. The fos and collagenase mRNA can be detected using therespective cDNA probes and analyzed by Northern blot analysis. Thisallows the determination of agents capable of inhibiting collagenaseproduction and an approximation of the step in the collagenase syntheticpathway that is affected by the treatment.

A. Materials and Methods

1. Effect of Paclitaxel on c-fos Expression

Chondrocytes were treated with different concentrations of paclitaxel(10⁻⁶ M, 10⁻⁷ M, and 10⁻⁸ M) for 2 hours and then treated with TNFa(Sigma Chemical Co., St. Louis, Mo.) at 30 ng/ml for 1 hour. Humanrecombinant TNFα was dissolved in phosphate buffered saline (PBS) with0.1% bovine serum albumin (BSA). Total RNA from bovine articularchondrocytes was isolated by the acidified guanidine isothiocyanatemethod and the levels of c-fos mRNA determined by Northern blotanalysis. Denatured RNA samples (12 μg) were analyzed by gelelectrophoresis in a denaturing 1% agarose gel, transferred to a nylonmembrane (Bio-Rad), cross-linked with an ultraviolet cross-linker(Stratagene UV stratalinker 1800), and hybridized with ³²P-labeled ratc-fos DNA. mRNA for tubulin and total RNA were used as controls. Todetermine tubulin mRNA, the blots described above were subsequentlystripped of DNA and re-probed with ³²P-labeled rat P-tubulin cDNA. Thisexperimented was conducted three times and the data collated.

2. Effect of Paclitaxel on Collagenase Expression

Chondrocytes were treated with different concentrations of paclitaxel(10⁻⁶ M and 10⁻⁷ M,) for 2 hours prior to the addition of IL-1 (20ng/ml). The cells were then incubated for a further 16 hours. Total RNAfrom bovine articular chondrocytes was isolated by the acidifiedguanidine isothiocyanate method and the collagenase mRNA determined byNorthern blot analysis. The RNA samples were prepared as described aboveusing ³²P-labeled rat collagenase cDNA.

B. Results

1. Effect of Paclitaxel on c-fos Expression

This experiment demonstrates that paclitaxel does not alter c-fosexpression at any concentration (see FIG. 36). Comparable levels ofc-fos mRNA were detectable in the controls and all of the experimentalgroups regardless of the paclitaxel concentration present. Total RNA andtubulin expression was similarly unaffected.

2. Effect of Paclitaxel on Collagenase Expression

This experiment demonstrates that paclitaxel at a concentration of 10⁻⁶M completely inhibited IL-1 induced collagenase expression. CollagenasemRNA was not detectable above background at this concentration ofpaclitaxel in vitro (see FIG. 37).

C. Discussion

Paclitaxel is capable of inhibiting collagenase production bychondrocytes in vitro at concentrations of 10⁻⁶M. This inhibition occursdownstream from the transcription factor activity of c-fos, but stillrepresents a secondary gene response, as collagenase mRNA production isaffected. As such, paclitaxel inhibition of collagenase production isnot strictly due to interruption of the microtubules involved in theprotein secretory pathway (which is dependent upon microtubular functionfor the movement of secretory granules), but acts at the level of thegene response to stimulation of collagenase production. Regardless ofthe mechanism of action, paclitaxel is capable of inhibiting collagenaseproduction by at least one cell type known to produce this enzyme in thearthritic disease process.

Example 25 Effect of Paclitaxel on Chondrocyte Viability

While it is important that a disease modifying agent be capable ofstrongly inhibiting a variety of inappropriate cellular activities(proliferation, inflammation, proteolytic enzyme production) which occurin excess during the development of RA, it must not be toxic to thenormal joint tissue. It is particularly critical that normalchondrocytes not be damaged, as this would hasten the destruction of thearticular cartilage and lead to progression of the disease. In thisexample, the effect of paclitaxel on normal chondrocyte viability invitro was examined.

Briefly, chondrocytes were incubated in the presence (10⁻⁵ M, 10⁻⁷ M,and 10⁻⁹ M) or absence (control) of paclitaxel for 72 hours. At the endof this time period, the total number of viable chondrocytes wasdetermined visually by dye exclusion counting using Trypan bluestaining. This experiment was conducted 4 times and the data collated.

Results of this experiment are shown in FIG. 38. Briefly, as is evidentfrom FIG. 38, paclitaxel does not affect the viability of normalchondrocytes in vitro even at high concentrations (10⁻⁵ M) ofpaclitaxel. More specifically, even at drug concentrations sufficient toblock the pathological processes described in the preceding examples,there is no cytotoxicity to normal chondrocytes.

Example 26 Ophthalmic Drops Containing Paclitaxel or PrednisoloneAcetate

Three formulations containing 0.3% paclitaxel for ophthalmic use wereprepared. The particle size distribution of paclitaxel as received froma supplier was not within acceptable limits for ophthalmic use. Inparticular, for ophthalmic drops at least 90% of the particles shouldpreferably be below 10 μm, with no particle above 20 μm. Two methodswere used to reduce the particle size. The first method involvedprecipitating paclitaxel from its solution in acetone. Briefly, 150 mgof paclitaxel was dissolved in 5 ml of acetone. This solution was addedin a gentle stream, with stirring, to 20 ml of Sterile water USP toprecipitate the drug. The suspension was homogenized with the Douncehomogenizer until about 90% of the drug was under 10 μm. The suspensionwas allowed to stand for about 1 hour. The larger particles settled andwere separated from the smaller ones by decantation. The largerparticles were again reduced until all particles were under 20 μm (seeFIG. 48). This suspension was added to the one previously decanted andthe acetone was evaporated by heating at 50° C. for 2 hours and then ina vacuum oven at 30° C. and 25 torr overnight to remove the residualacetone. Sodium chloride (0.45 g) was dissolved in 5 ml Sterile waterUSP. This solution and 20 ml of 5% PVA solution were mixed with thepaclitaxel suspension, made up to 50 ml with sterile water and bottled.

Paclitaxel suspension (0.3%) was also prepared by adding 150 mg ofaclitaxel to 10 ml of sterile water and comminuted using the FritschPulverizer for 15 minutes. It was not possible to produce particleslower than 60 μm with this method probably because the solid paclitaxelwas not hard and brittle (see FIG. 49). The suspension was mixed with 5ml sterile water containing 0.45 g of NaCl and 20 ml of 5% PVA solutionand made up to 50 ml with sterile water.

Paclitaxel microspheres containing 10% paclitaxel in PCL were alsoprepared. Briefly, paclitaxel (60 mg) and PCL (540 mg) were dissolved in3 ml of DCM, 20 ml of 3% PVA solution was added and homogenized with thePolytron homogenizer at point 3 setting for about 1 minute. The emulsionwas poured into a 30 ml beaker and stirred until the microspheres wereformed (about 3 hours). This suspension was placed in a vacuum oven, at30° C. and 25 torr, overnight to remove residual DCM. The smallmicrosphere suspension (15 ml) was decanted and evaporated under vacuumto about 5 ml and assayed for paclitaxel. This suspension was mixed with2 ml solution of NaCl (0.45 g) solution and made up to 10 ml withsterile water.

Prednisolone acetate suspension containing 1% drug was prepared byhomogenizing the appropriate amount of the drug (as received) in 20 mlof 5% PVA, NaCl solution was added and made up to volume with sterilewater.

Example 27 Paclitaxel in an Animal Model of Corneal Neovascularization

Induction of Corneal Neovascularization

Corneal angiogenesis is induced in male New Zealand white rabbits (2.5to 3.0 kg) essentially as described by Scroggs et al., Invest.Ophthalmol. Vis. Sci. 32:2105-2111, 1991. Briefly, rabbits areanesthetized with a subcutaneous injection of 0.15 cc of a 1:1 mixtureof ketamine (80 mg/ml) and xylazine (4 mg/ml), and the eyes cauterizedby applying the tip of a new silver-potassium nitrate applicator (75%silver nitrate: 25% potassium nitrate; Graham-Field, Hauppage, N.Y.) 3to 4 millimeters from the corneo-scleral limbus.

Immediately following chemical cauterization, one drop of the studysolution (e.g., the study solutions may be vehicle alone, prednisoloneacetate 1%, or 0.3% paclitaxel in suspension) is applied to thecauterized eyes. Gentamicin ophthalmic ointment is then applied to thetreated eyes. Over the next two weeks, one drop of the study solution isapplied four times daily.

In a second study, 0.5 ml aliquots of a 10% paclitaxel-loadedmicrosphere suspension and a 20% paclitaxel-loaded thermopaste isadministered via subconjuctival injection to the experimental animals.

On the eighth day and fourteenth following cauterization of the corneas,all animals are re-anesthetized as described above, and the corneasphotographed using a Nikon biomicroscope and Kodak ASA 180 tungsten filmunder microscope incandescent illumination. The highest magnificationthat incorporates the entire cornea is used.

The photographs are randomly presented to a masked observer who gradesthe corneal vessels based upon a 0 to 4 scale of vessel density, and whomeasures the total extent in clock hours of circumferential cornealneovascularization. Vessel density grade is based on two standardphotographs obtained from pilot experiments that had been assignedgrades 2 (moderate vessel density) and 4 (severe vessel density)respectively. Grades 1 and 3 were established be interpolation; grade 0is applied to corneas that demonstrate a central cautery scar, but theabsence of new vessel growth.

Differences in both comeal vessel density and extent, in terms of clockhours of involvement, is analyzed using non-paired Student's t tests.Tests are two-tailed, with a p value of ≦0.05 considered significant.Measures are reported as mean±standard deviation.

Example 28 Modification of Paclitaxel Release from Thermopaste using LowMolecular Weight Poly(D,L, Lactic Acid)

As discussed above, depending on the desired therapeutic effect, eitherquick release or slow release polymeric carriers may be desired. Forexample, polycaprolactone (PCL) and mixtures of PCL with poly(ethyleneglycol) (PEG) produce compositions which release paclitaxel over aperiod of several months. In particular, the diffusion of paclitaxel inthe polymers is very slow due to its large molecular size and extremehydrophobicity.

On the other hand, low molecular weight poly(DL-lactic acid) (PDLLA)gives fast degradation, ranging from one day to a few months dependingon its initial molecular weight. The release of paclitaxel, in thiscase, is dominated by polymer degradation. Another feature of lowmolecular weight PDLLA is its low melting temperature, (i.e., 40° C.-60°C.), which makes it suitable material for making Thermopaste. Asdescribed in more detail below, several different methods can beutilized in order to control the polymer degradation rate, including,for example, by changing molecular weight of the PDLLA, and/or by mixingit with high mol wt. PCL, PDLLA, or poly(lactide-co-glyocide) (PLGA).

A. Experimental Materials

D,L-lactic acid was purchased from Sigma Chemical Co., St. Louis, Mo.PCL (molecular weight 10-20,000) was obtained from Polysciences,Warrington, Pa. High molecular weight PDLLA (intrinsic viscosity 0.60dl/g) and PLGA (50:50 composition, viscosity 0.58 dl/g) were fromBirmingham Polymers.

B. Synthesis of Low Molecular weight PDLLA

Low molecular weight PDLLA was synthesized from DL-lactic acid throughpolycondensation. Briefly, DL-lactic acid was heated in a glass beakerat 200° C. with nitrogen purge and magnetic stirring for a desired time.The viscosity increased during the polymerization, due to the increaseof molecular weight. Three batches were obtained with differentpolymerization times, i.e., 40 min (molecular weight 800), 120 min, 160min.

C. Formulation of Paclitaxel Thermopastes

Paclitaxel was loaded, at 20%, into the following materials by handmixing at a temperature about 60° C.

1. low molecular weight PDLLA with polymerization time of 40 min.

2. low molecular weight PDLLA with polymerization time of 120 min.

3. low mol. wt PDLLA with polymerization time of 160 min.

4. a mixture of 50:50 high molecular weight PDLLA and low molecularweight PDLLA 40 min.

5. a mixture of 50:50 high molecular weight PLGA and low molecularweight PDLLA 40 min.

6. mixtures of high molecular weight PCL and low molecular weight. PDLLA40 min with PCL:PDLLA of 10:90, 20:80, 40:60, 60:40, and 20:80. Mixturesof high molecular weight PDLLA or PLGA with low molecular weight. PDLLAwere obtained by dissolving the materials in acetone followed by drying.

D. Release Study

The release of paclitaxel into PBS albumin buffer at 37° C. was measuredas described above with HPLC at various times.

E. Results

Low molecular weight PDLLA 40 min was a soft material with light yellowcolor. The color is perhaps due to the oxidation during thepolycondensation. Low molecular weight PDLLA 120 min (yellow) and 160min (brown) were brittle solids at room temperature. They all becomemelts at 60° C. Mixtures of 50:50 high molecular weight PDLLA or PLGAwith low molecular weight PDLLA 40 min also melted about 60° C.

During the release, low molecular weight PDLLA 40 min and 120 min brokeup into fragments within one day, other materials were intact up to thiswriting (3 days).

The release of paclitaxel from formulations 2-5 were shown in FIG. 50.Low molecular weight PDLLA 40 min and 120 min gave the fastest releasedue to the break up of the paste. The release was perhaps solubilitylimited. Low molecular weight PDLLA 160 min. also gave a fast releaseyet maintained an intact pellet. For example, 10% of loaded paclitaxelwas released with one day. The 50:50 mixtures of high molecular weightPDLLA or PLGA with low molecular weight PDLLA 40 min were slower, i.e.,3.4% and 2.2% release within one day.

Although not specifically set forth above, a wide variety of otherpolymeric carriers may be manufactured, including for example, (1) lowmolecular weight (500-10,000) poly(D,L-lactic acid), poly(L-lacticacid), poly(glycolic acid), poly(6-hydroxycaproic acid),poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), and theircopolymers; (2) blends of above (#1) above; (3) blends of (#1) abovewith high molecular weight poly(DL-lactic acid), poly(L-lactic acid),poly(glycolic acid), poly(6-hydroxycaproic acid), poly(5-hydroxyvalericacid), poly(4-hydroxybutyric acid), and their copolymers; and (4)copolymers of poly(ethylene glycol) and pluronics with poly(D,L-lacticacid), poly(L-lactic acid), poly(glycolic acid), poly(6-hydroxycaproicacid), poly(5-hydroxyvaleric acid), poly(4-hydroxybutyric acid), andtheir copolymers.

Example 29 Surfactant Coated Microspheres

A. Materials and Methods

Microspheres were manufactured from Poly (DL) lactic acid (PLA), polymethylmethacrylate (PMMA), polycaprolactone (PCL) and 50:50 Ethylenevinyl acetate (EVA):PLA essentially as described in Example 8. Sizeranged from 10 to 100 um with a mean diameter 45 um.

Human blood was obtained from healthy volunteers. Neutrophils (whiteblood cells) were separated from the blood using dextran sedimentationand Ficoll Hypaque centrifugation techniques. Neutrophils were suspendedat 5 million cells per ml in Hanks Buffered Salt Solution (“HBSS”).

Neutrophil activation levels were determined by the generation ofreactive oxygen species as determined by chemiluminescence. Inparticular, chemiluminescence was determined by using an LKB luminometerwith 1 uM luminol enhancer. Plasma precoating (or opsonization) ofmicrospheres was performed by suspending 10 mg of microspheres in 0.5 mlof plasma and tumbling at 37° C. for 30 min.

Microspheres were then washed in 1 ml of HBSS and the centrifugedmicrosphere pellet added to the neutrophil suspension at 37° C. at time.t=0. Microsphere surfaces were modified using a surfactant calledPluronic F127 (BASF) by suspending 10 mg of microspheres in 0.5 ml of 2%w/w solution of F127 in HBSS for 30 min at 37° C. Microspheres were thenwashed twice in 1 ml of HBSS before adding to neutrophils or to plasmafor further precoating.

B. Results

FIG. 51 shows that the untreated microspheres give chemiluminescencevalues less than 50 mV. These values represent low levels of neutrophilactivation. By way of comparison, inflammatory microcrystals might givevalues close to 1000 mV, soluble chemical activators might give valuesclose to 5000 mV. However, when the microspheres are precoated withplasma, all chemiluminescence values are amplified to the 100 to 300 mVrange (see FIG. 51). These levels of neutrophil response or activationcan be considered mildly inflammatory. PMMA gave the biggest responseand could be regarded as the most inflammatory. PLA and PCL both becomethree to four times more potent in activating neutrophils after plasmapretreatment (or opsonization) but there is little difference betweenthe two polymers in this regard. EVA:PLA is not likely to be used inangiogenesis formulations since the microspheres are difficult to dryand resuspend in aqueous buffer. This effect of plasma is termedopsonization and results from the adsorption of antibodies or complementmolecules onto the surface. These adsorbed species interact withreceptors on white blood cells and cause an amplified cell activation.

FIGS. 52-55 describe the effects of plasma precoating of PCL, PMMA, PLAand EVA:PLA respectively as well as showing the effect of pluronic F127precoating prior to plasma precoating of microspheres. These figures allshow the same effect: (1) plasma precoating amplifies the response; (2)Pluronic F127 precoating has no effect on its own; (3) the amplifiedneutrophil response caused by plasma precoating can be stronglyinhibited by pretreating the microsphere surface with 2% pluronic F127.

The nature of the adsorbed protein species from plasma was also studiedby electrophoresis. Using this method, it was shown that pretreating thepolymeric surface with Pluronic F127 inhibited the adsorption ofantibodies to the polymeric surface.

FIGS. 56-59 likewise show the effect of precoating PCL, PMMA, PLA orEVA:PLA microspheres (respectively) with either IgG (2 mg/ml) or 2%pluronic F127 then IgG (2 mg/ml). As can be seen from these figures, theamplified response caused by precoating microspheres with IgG can beinhibited by treatment with pluronic F127.

This result shows that by pretreating the polymeric surface of all fourtypes of microspheres with Pluronic F127, the “inflammatory” response ofneutrophils to microspheres may be inhibited.

Example 30 Preparation of Low Molecular Weight Poly(D,L-Lactic Acid)

Five hundred grams of D,L-lactic acid (Sigma Chemical Co., St. Louis,Mo.) was heated in a heating mantle at 190° C. for 90 minutes under astream of nitrogen gas. This process produced 400 g of poly(D,L-lacticacid) with a molecular weight of 700-800 as determined by end grouptitration and gel permeation chromatography (Fukusaki et al, Eur. Polym.J. 25(10):1019-1026, 1989).

Example 31 Preparation of Polymeric Compositions Containing GelatinizedPaclitaxel

A. Preparation of Polymers

Two hundred milligrams of gelatin (Type B, bloom strength 225, FisherScientific) 200 mg of NaCl, or 100 mg of gelatin and 100 mg of NaCl weredissolved in 0.5 mL of water. Next, 200 mg of paclitaxel was dissolvedin 0.5 mL of ethanol. The dissolved gelatin, salt, or gelatin and saltwere then added to the paclitaxel and triturated on a petri dishincubating in a water bath at 80° C., until dry. The precipitate wasthen ground in a mortar and pestle and sieved through either no. 60 orno. 140 mesh (Endecott, London, England). (No. 60 mesh produces largergranules and no. 140 mesh produces smaller granules.)

Polycaprolactone was then heated to 60° C., and granules added to afinal ratio of 40:60 (w/w). The polymeric composition was placed into a1 ml syringe and extruded.

B. Analysis of Paclitaxel Release

A measured amount of the cylindrical polymeric composition is then addedinto an albumin buffered solution, and, over a time course, aliquots areremoved and paclitaxel extracted with DCM. The extracts are thenanalyzed by HPLC. Results of these experiments are shown in FIGS. 39 and40. Briefly, FIG. 38 shows a greater percentage of paclitaxel releasedwhen large gelatinized particles (>200 μm) are utilized. FIG. 39 showsthat addition of NaCl is not preferred when higher amounts of paclitaxelrelease is desired.

Example 32 Copolymerization of Poly(D,L-Lactic Acid) and PolyethyleneGlycol

D,L,lactide (Aldrich Chemical Co.) was added to polyethylene glycol(molecular weight 8,000; Sigma Chemical Co., St. Louis, Mo.) in a tubeand heated with 0.5% stannous octoate (Sigma Chemical Co.) for 4 hoursat 150° C. in an oven.

This process produces a copolymer of poly(D,L-lactic acid) withpolyethylene glycol as a triblock polymer (i.e., PDLLA-PEG-PDLLA).Paclitaxel release from this polymer is shown in FIG. 41.

Example 33 Analysis of Drug Release

A known weight of a polymer (typically a 2.5 mg pellet) is added to a 15ml test tube containing 14 ml of a buffer containing 10 mmNa₂HPO₄—NaH₂PO₄, 0.145 m NaCl and 0.4 g/l bovine serum albumin. Thetubes are capped and tumbled at 37° C. At specific times all the 14 mlof the liquid buffer are removed and replaced with fresh liquid buffer.

The liquid buffer is added to 1 milliliter of methylene chloride andshaken for 1 minute to extract all the paclitaxel into the methylenechloride. The aqueous phase is then removed and the methylene chloridephase is dried under nitrogen. The residue is then dissolved in 60%acetonitrile: 40% water and the solution is injected on to a HPLC systemusing the following conditions: C8 column (Beckman Instruments USA),mobile phase of 58%:5%:37% acetonitrile: methanol: water at a flow rateof 1 minute per minute.

For paclitaxel the collected buffer is then analyzed at 232 nm. For MTXthe collected buffer is applied directly to the HPLC column with no needfor extraction in methylene chloride. MTX is analyzed at 302 nm. ForVanadium containing compounds the liquid buffer is analyzed directlyusing a UV/VIS spectrometer in the 200 to 300 nm range.

Example 34 Manufacture of Polymeric Compositions Containing PCL andMePEG

A. Paclitaxel Release from PCL

Polycaprolactone containing various concentrations of paclitaxel wasprepared as described in Example 10. The release of paclitaxel over timewas measured by HPLC essentially as described above. Results are shownin FIG. 42.

B. Effect of MePEG on Paclitaxel Release

MePEG at various concentrations was formulated into PCL paste containing20% paclitaxel, utilizing the methods described in Example 10. Therelease of paclitaxel over time was measured by HPLC essentially asdescribed above. Results of this study are shown in FIG. 43.

C. Effect of MePEG on the Melting Point of PCL

MePEG at various concentrations (formulated into PCL paste containing20% paclitaxel) was analyzed for melting point using DSC analysis at aheating rate of 2.5° C. per minute ; Results are shown in FIG. 44A(melting point vs. % MePEG) and 44B (percent increase in time tosolidify vs. % MePEG).

D. Tensile Strength of MePEG Containing PCL

PCL containing MePEG at various concentrations was tested for tensilestrength and time to fail by a CT-40 Mechanical Strength Tester. Resultsare shown in FIG. 45.

E. Effect of γ-irradiation or the Release of Paclitaxel

PCL:MePEG (80:20) paste loaded with 20% paclitaxel was γ-irradiated andanalyzed for paclitaxel release over time. Results are set forth in FIG.46.

In summary, based on the above experiments it can be concluded that theaddition of MePEG makes the polymer less brittle and more wax like,reduces the melting point and increases the solidification time of thepolymer. All these factors improve the application properties of thepaste. At low concentrations (20%) MePEG has no effect on the release ofpaclitaxel from PCL. Gamma-irradiation appears to have little effect onpaclitaxel release.

Example 35 Methotrexate-Loaded Paste

A. Manufacture of Methotrexate-Loaded Paste

Methotrexate (“MTX”; Sigma Chemical Co.) is ground in a pestle andmortar to reduce the particle size to below 5 microns. It is then mixedas a dry powder with polycaprolactone (molecular wt 18000 BirminghamPolymers, AL USA). The mixture is heated to 65° C. for 5 minutes and themolten polymer/methotrexate mixture is stirred into a smooth paste for.5 minutes. The molten paste is then taken into a 1 mL syringe, andextruded as desired.

B. Results

Results are shown in FIGS. 47A-E. Briefly, FIG. 47A shows MTX releasefrom PCL discs containing 20% MePEG and various concentrations of MTX.FIG. 47B shows a similar experiment for paste which does not containMePEG. FIGS. 47C, D, and E show the amount of MTX remaining in the disk.

As can be seen by the above results, substantial amounts of MTX can bereleased from the polymer when high MePEG concentrations are utilized.

Example 36 Manufacture of Microspheres Containing Methotrexate

A. Microspheres With MTX Alone

Methotrexate (Sigma) was ground in a pestle and mortar to reduce theparticle size to below 5 microns. One hundred milliliters of a 2.5% PVA(w/v) (Aldrich or Sigma) in water was stirred for 15 minutes with 500 mgof unground MTX at 25° C. to saturate the solution with MTX. Thissolution was then centrifuged at 2000 rpm to remove undissolved MTX andthe supernatant used in the manufacture of microspheres.

Briefly, 10 ml of a 5% w/v solution of poly(DL) lactic acid (molecularweight 500,000; Polysciences), Polylactic:glycolic acid (50:50 IV 0.78polysciences) or polycaprolactone (molecular weight 18,000, BPI)containing 10:90 w/w MTX(ground):POLYMER were slowly dripped into 100 mLof the MTX saturated 2.5% w/v solution of PVA (Aldrich or Sigma) withstirring at 600 rpm. The mixture was stirred at 25° C. for 2 hours andthe resulting microspheres were washed and dried.

Using this method MTX loaded microspheres can be reproduciblymanufactured in the 30 to 160 micron size range.

FIG. 60 depicts the results for 10% methotrexate-loaded microspheresmade from PLA:GA (50:50); Inherent Viscosity “IV”=0.78.

B. Microspheres With MTX and Hyaluronic Acid

MTX loaded microspheres can be made using hyaluronic acid (“HA”) as thecarrier by a water in oil emulsion manufacture method, essentially asdescribed below. Briefly, 50 ml of Parafin oil (light oil; FisherScientific) is warmed to 60° C. with stirring at 200 rpm. A 5 mLsolution of sodium hyaluronate (20/mL); source=rooster comb; Sigma) inwater containing various amounts MTX is added dropwise into the Parafinoil. The mixture is stirred at 200 rpm for 5 hours, centrifuged at 500×gfor 5 minutes. The resulting microspheres are washed in hexane fourtimes, and allowed to dry.

Example 37 Manufacture of Polymeric Compositions Containing VanadiumCompounds

A. Polymeric Paste Containing Vanadyl Sulfate

Vanadyl Sulfate (Fisher Scientific) is first ground in a pestle andmortar to reduce the particle size, then dispersed into melted PCL asdescribed above for MTX. It is then taken up into a syringe to solidifyand is ready for use.

Drug release was determined essentially as described above in Example33, except that a 65 mg pellet of a 10% w/w VOSO₄:PCL was suspended in10 ml of water and the supernatant analyzed for released VanadylSulphate using UV/V is absorbance spectroscopy of the peak in the 200 to300 nm range.

Results are shown in FIG. 61. Briefly, from a polymeric compositioncontaining 10% VOSO₄, 1 mg of VOSO₄ was released in 6 hours, 3 mg after2 days and 5 mg by day 6.

B. Polymeric Microspheres Containing Vanadyl Sulfate

Vanadyl sulfate was incorporated into microspheres of polylactic acid orhyaluronic acid essentially as described in Example 36B. Results areshown in FIG. 62.

C. Polymeric Paste Containing Organic Vanadate

Organic vanadate is loaded into a PCL paste essentially as describedabove in Example 35. Vanadate release from the microspheres wasdetermined as described above and in Example 33. Results are shown inFIGS. 63A and 63B.

D. Organic Vanadate Containing Microspheres

Organic vanadate may also be loaded into microspheres essentially asdescribed in Example 36A. Such microspheres are shown in FIG. 64 forpoly D,L lactic acid (M.W. 500,000; Polysciences).

Example 38 Polymeric Compositions with Increased Concentrations ofPaclitaxel

PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with hydrophobic(PDLLA) and hydrophilic (PEG or MePEG) regions. At appropriate molecularweights and chemical composition, they may form tiny aggregates ofhydrophobic PDLLA core and hydrophilic MePEG shell. Paclitaxel can beloaded into the hydrophobic core, thereby providing paclitaxel with anincreased “solubility”.

A. Materials

D,L-lactide was purchased from Aldrich, Stannous octoate, poly (ethyleneglycol) (mol. wt. 8,000), MePEG (mol. wt. 2,000 and 5,000) were fromSigma. MePEG (mol. wt. 750) was from Union Carbide. The copolymers weresynthesized by a ring opening polymerization procedure using stannousoctoate as a catalyst (Deng et al, J. Polym. Sci., Polym, Lett.28:411-416, 1990; Cohn et al, J. Biomed, Mater. Res. 22: 993-1009,1988).

For synthesizing PDLLA-MePEG, a mixture of DL-lactide/MePEG/stannousoctoate was added to a 10 milliliter glass ampoule. The ampoule wasconnected to a vacuum and sealed with flame. Polymerization wasaccomplished by incubating the ampoule in a 150° C. oil bath for 3hours. For synthesizing PDLLA-PEG-PDLLA, a mixture ofD,L-lactide/PEG/stannous octoate was transferred into a glass flask,sealed with a rubber stopper, and heated for 3 hours in a 150° C. oven.The starting compositions of the copolymers are given in Tables V andVI. In all the cases, the amount of stannous octoate was 0.5%-0.7%.

B. Methods

The polymers were dissolved in acetonitrile and centrifuged at 10,000 gfor 5 minutes to discard any non-dissolvable impurities. Paclitaxelacetonitrile solution was then added to each polymer solution to give asolution with paclitaxel (paclitaxel+polymer) of 10%-wt. The solventacetonitrile was then removed to obtain a clear paclitaxel/PDLLA-MePEGmatrix, under a stream of nitrogen and 60° C. warning. Distilled water,0.9% NaCl saline, or 5% dextrose was added at four times weight of thematrix. The matrix was finally “dissolved” with the help of vortexmixing and periodic warming at 60° C. Clear solutions were obtained inall the cases. The particle sizes were all below 50 mn as determined bya submicron particle sizer, NICOMP Model 270. The formulations are givenin Table V. TABLE V Formulations of Paclitaxel/PDLLA-MePEG* PaclitaxelLoading PDLLA-MePEG Dissolving Media (final paclitaxel concentrate)2000/50/50 water 10% (20 mg/ml) 2000/40/60 water 10% (20 mg/ml)2000/50/50 0.9% saline  5% (10 mg/ml) 2000/50/50 0.9% saline 10% (20mg/ml) 2000/50/50 5% dextrose 10% (10 mg/ml) 2000/50/50 5% dextrose 10%(20 mg/ml)*PEG molecular weight. 8,000.

In the case of PDLLA-PEG-PDLLA, since the copolymers cannot dissolve inwater, paclitaxel and the polymer were co-dissolved in acetone. Water ora mixture of water/acetone was gradually added to this paclitaxelpolymer solution to induce the formation of paclitaxel/polymer spheres.TABLE VI Composition of PDLLA-PEG-PDLLA Copolymer Name Wt. of PEG (g)Wt. of DL-lactide (g) PDLLA-PEG-PDLLA 1 9 90/10 PDLLA-PEG-PDLLA 2 880/20 PDLLA-PEG-PDLLA 3 7 70/30 PDLLA-PEG-PDLLA 4 6 60/40PDLLA-PEG-PDLLA 14 6 30-/70C. Results

Many of the PDLLA-MePEG compositions form clear solutions in water, 0.9%saline, or 5% dextrose, indicating the formation of tiny aggregates inthe range of nanometers. Paclitaxel was loaded into PDLLA-MePEGnanoparticles successfully. For example, at % loading (this represents10 mg paclitaxel in 1 ml paclitaxel/PDLLA-MePEG/aqueous system), a clearsolution was obtained from 2000-50/50 and 2000-40/60. The particle sizewas about 20 nm.

Example 39 Insertion of Control and Paclitaxel Coated Stents intoMicroswine

As discussed above, various tubes within the body can be occluded bydisease processes. One method for treating such occlusion is to insertan endoluminal stent within the tube in order to relieve theobstruction. Unfortunately, the stents themselves are often overgrown byepithelial cells, thus limiting the duration and effectiveness of thetreatment. As described in more detail below, stainless steel stentswere coated with paclitaxel-loaded EVA polymer and placed into thebiliary duct of microswine in order to assess prevention of benignepithelial overgrowth.

A. Materials and Methods

Yucatan microswine were placed under general anesthetic and a 5 cmtransverse upper abdominal incision performed. The gallbladder wasgrasped and sewn to the anterior abdominal wall and a tiny incision wasmade in the gallbladder fundus. A 5 F catheter was inserted into thegallbladder and radiopaque contrast injected to outline the biliarytree. A hydrophilic guidewire was advanced through the cystic duct intothe common bile duct, and over this a 7 F (purpose—built, reusable)delivery catheter containing a stainless steel (5 mm diameter×4.2 cmlong) was advanced and deployed in the common bile duct. The deliverycatheter was withdrawn into the gallbladder and a repeat cholangiogramperformed. The gallbladder incision was closed, a radiopaque staple wasfixed at the incision site, and then the abdominal incision was closed.The swine was randomized into groups of receiving uncoated stents,polymer coated stents, and paclitaxel-loaded (33%) polymer coatedstents. Tantalum (strecker) stainless steel stents and stainless steelWallstents were used for each of these studies. Swines from each groupwere sacrificed at 14, 28, 56, and 112 days post-stent insertion byinjecting Euthanyl. After sacrifice, the gallbladder was cannulatedpercutaneously under X-ray by puncturing at the staple and radiopaquecontrast injected to outline the biliary tree. X-rays were taken andanalyzed for narrowing at or adjacent to the stent. The liver andbiliary tree were removed en bloc. The portion of bile duct containingthe stent was sectioned transversely at 1 cm intervals and thehistologic sections were used to assess the degree of overgrowth of thestent. The liver was also examined histologically for signs of chronicobstruction or inflammation.

B. Results

Control, uncoated stainless steel stents were inserted into microswineas described above, and sacrificed at various times. At two weeks, thebile mucosa appeared normal in 2 of the sacrificed pigs, while onepresented a small non-obstructive bile concretion within the biliarylumen, and a slight indentation in the bile duct mucosa at the site ofthe tines. At 4 weeks, of the 3 pigs which were sacrificed a small bileconcretion was present on the distal stent, as well as mucosalindentations of the stent tine within the bile duct mucosa. At 8 weeks,the bile duct. mucosa at the site of stent insertion in the sacrificedpigs partially overgrew the stent tines in a crescentic manner overapproximately 25-30% of the radius of the stent (FIGS. 65A, 66A, and66B). In addition, one pig contained thick bile containing inflammatorycells within the lumen. At 16 weeks, pigs which were sacrificedpresented a stent which was completely overgrown distally by fibroustissue, and no evidence of a lumen (FIG. 65B). Histologically, thetissue was uniformly fibrous. Surprisingly, the liver biopsy of all ofthe control treated swines were normal and there was no evidence ofobstructive changes.

In another group of microswine, stents coated with ethylene vinylacetate and 33% paclitaxel were inserted into the biliary duct. After an8 week exposure, one pig was sacrificed and showed a slight indentationof the bile duct mucosa at the site of the stent tines and no indicationof overgrowth (FIGS. 66C and 66D). The underlying mucosa was normalapart from some inflammatory cell infiltration. Non-obstructive bileconcretions were noted in the lumen of the stent. The liver biopsy wasnormal, with no evidence of obstructive changes.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A composition comprising: (a) an anti-angiogenic factor; and (b) apolymeric carrier.
 2. The composition of claim 1, wherein saidanti-angiogenic factor is Anti-Invasive Factor.
 3. The composition ofclaim 1, wherein said anti-angiogenic factor is retinoic acid andderivatives thereof.
 4. The composition of claim 1, wherein saidanti-angiogenic factor is selected from the group consisting of Suramin,Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor ofMetalloproteinase-2, Plasminogen Activator Inhibitor-1 and PlasminogenActivator Inhibitor-2.
 5. A composition comprising: (a) taxol; and (b) apolymeric carrier.
 6. The composition of claim 1, wherein saidcomposition has an average size of 15 to 200 lm.
 7. The composition ofclaim 1, wherein said polymeric carrier is poly(ethylene-vinyl acetate)crosslinked with 40% vinyl acetate.
 8. The composition of claim 1,wherein said polymeric carrier is poly(lactic-co-glycolic acid).
 9. Thecomposition of claim 1, wherein said polymeric carrier ispolycaprolactone.
 10. The composition of claim 1, wherein said polymericcarrier is polylactic acid.
 11. The composition of claim 1, wherein saidpolymeric carrier is a copolymer of poly(ethylene-vinyl acetate)crosslinked with 40% vinyl acetate, and polylactic acid.
 12. Thecomposition of claim 1, wherein said polymeric carrier is a copolymer ofpolylactic acid and polycaprolactone.
 13. A method for embolizing ablood vessel, comprising delivering into said vessel a therapeuticallyeffective amount of composition according to claims 1-12, such that saidblood vessel is effectively occluded.
 14. The method claim 13 whereinsaid blood vessel nourishes a tumor.
 15. A stent, comprising a generallytubular structure, the surface of which is coated with a compositionaccording to claims 1-12.
 16. A method for expanding the lumen of a bodypassageway, comprising inserting a stent into the passageway, the stenthaving a generally tubular structure, the surface of said structurebeing coated with a composition according to claims 1-12, such that saidpassageway is expanded.
 17. A method for eliminating vascularobstructions, comprising inserting a vascular stent into a vascularpassageway, the stent having a generally tubular structure, the surfaceof said structure being coated with a composition according to claims1-12, such that said vascular obstruction is eliminated.
 18. A methodfor eliminating biliary obstructions, comprising inserting a biliarystent into a biliary passageway, the stent having a generally tubularstructure, the surface of said structure being coated with a compositionaccording to claims 1-12, such that said biliary obstruction iseliminated.
 19. A method for eliminating urethral obstructions,comprising inserting a urethral stent into a urethra, the stent having agenerally tubular structure, the surface of said structure being coatedwith a composition according to claims 1-12, such that said urethralobstruction is eliminated.
 20. A method for eliminating esophagealobstructions, comprising inserting an esophageal stent into anesophagus, the stent having a generally tubular structure, the surfaceof said structure being coated with a composition according to claims1-12, such that said esophageal obstruction is eliminated.
 21. A methodfor eliminating tracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi, the stent having agenerally tubular structure, the surface of which is coated with acomposition according to claims 1-12, such that said tracheal/bronchialobstruction is eliminated.
 22. A method for treating a tumor excisionsite, comprising administering a composition according to claims 1-12 tothe resection margin of a tumor subsequent to excision, such that thelocal recurrence of cancer and the formation of new blood vessels atsaid site is inhibited.
 23. A method for treating comealneovascularization, comprising administering a therapeutically effectiveamount of a composition according to claims 1-12 to the cornea, suchthat the formation of blood vessels is inhibited.
 24. The method ofclaim 23, wherein said composition further comprises a topicalcorticosteroid.
 25. A method for inhibiting angiogenesis in patientswith non-tumorigenic, angiogenesis-dependent diseases, comprisingadministering a therapeutically effective amount of a compositioncomprising taxol to a patient with a non-tumorigenicangiogenesis-dependent disease, such that the formation of new bloodvessels is inhibited.
 26. A method for embolizing a blood vessel in anon-tumorigenic, angiogenesis-dependent diseases, comprising deliveringto said vessel a therapeutically effective amount of a compositioncomprising taxol, such that said blood vessel is effectively occluded.27. A method for expanding the lumen of a body passageway, comprisinginserting a stent into the passageway, the stent having a generallytubular structure, the surface of said structure being coated with acomposition comprising taxol, such that said passageway is expanded. 28.A method for eliminating vascular obstructions, comprising inserting avascular stent into a vascular passageway, the stent having a generallytubular structure, the surface of said structure being coated with acomposition comprising taxol, such that said vascular obstruction iseliminated.
 29. A method for eliminating biliary obstructions,comprising inserting a biliary stent into a biliary passageway, thestent having a generally tubular structure, the surface of saidstructure being coated with a composition comprising taxol, such thatsaid biliary obstruction is eliminated.
 30. A method for eliminatingurethral obstructions, comprising inserting a urethral stent into aurethra, the stent having a generally tubular structure, the surface ofsaid structure being coated with a composition comprising taxol, suchthat said urethral obstruction is eliminated.
 31. A method foreliminating esophageal obstructions, comprising inserting an esophagealstent into an esophagus, the stent having a generally tubular structure,the surface of said structure being coated with a composition comprisingtaxol, such that said esophageal obstruction is eliminated.
 32. A methodfor eliminating tracheal/bronchial obstructions, comprising inserting atracheal/bronchial stent into the trachea or bronchi, the stent having agenerally tubular structure, the surface of said structure being coatedwith a composition comprising taxol, such that said tracheal/bronchialobstruction is eliminated.
 33. A method for treating a tumor excisionsite, comprising administering a composition comprising taxol to theresection margin of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at said siteis inhibited.
 34. A method for treating corneal neovascularization,comprising administering a therapeutically effective amount of acomposition comprising taxol to the cornea, such that the formation ofnew vessels is inhibited.
 35. A pharmaceutical product, comprising: (a)taxol, in a container; and (b) a notice associated with said containerin form prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby said agency of said taxol, for human or veterinary administration totreat non-tumorigenic angiogenesis-dependent diseases.